The EMBO Journal Peer Review Process File - EMBO-2013-85162
Manuscript EMBO-2013-85162
Quantitative comparison of a Human Cancer Cell-Surface
Proteome between Interphase and Mitosis
Nurhan Özlü, Mohammad Haroon Qureshi, Yusuke Toyoda, Bernhard Y. Renard, Gürkan
Mollaoglu, Nazı Ezgi Özkan, Selda Bulbul, Ina Poser, Wiebke Timm, Anthony A. Hyman, Timothy
Mitchison, Judith Steen
Corresponding author: Nurhan Özlü, Koc University
Review timeline:
Submission date:
Editorial Decision:
Re-submission:
Editorial Decision:
Revision received:
Accepted:
27 March 2013
07 May 2013
03 February 2014
14 April 2014
25 August 2014
05 November 2014
Editor: Hartmut Vodermaier
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
07 May 2013
Thank you again for submitting your manuscript on mitotic cell surface proteomics for our
consideration at The EMBO Journal. We have in the meantime also received the third and last
report, and I have now been able to carefully consider all three reports, which you will find copied
below. Unfortunately, none of the three referees is currently strongly in favor of publication, as is
apparent from their reports but also reflected in the ratings and recommendations provided by the
reviewers. I am therefore very much afraid that we cannot offer to consider the manuscript further
for revision at this point. As you will see, even if we consider that referee 1 may have assessed the
manuscript from a somewhat too puristic biological viewpoint, it is apparent that s/he also does not
consider the current dataset of sufficiently compelling resource value for the field. On the other
hand, the two more technical reviewers have major concerns regarding a number of issues, including
methodological concerns, analysis and reporting of the obtained datasets, and the limited extent of
validation. Although I realize that you may well be able to address or clarify some of these points, I
hope you understand that we also have to take the overall enthusiasm and support from the referees
into account when deciding whether or not to invite a revision. Furthermore, as a broad general
journal we can only publish a limited number of dedicated Resource Articles, and therefore rather
strong endorsement of their value from both the biological and the methodological side would be
required to warrant publication in The EMBO Journal as opposed to a more methods- or proteomicsoriented journal. I am very sorry that we cannot be more positive on this occasion, but hope that you
will in any case find our referees' comments helpful.
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2013-85162
Referee #1
In this paper, Alper et al. describe a new method by which to assess differences in the protein
composition of the mitotic and interphase plasma membrane. This required the team to develop a
protocol in which isotope labeling together with amine and carboxylate chemistry are used to
differentially mark and purify biotinylated surface proteins in mitotic and interphase cells.
In this way the authors were then able to use MS to identify a long list of proteins that preferentially
accumulate on the cell surface in interphase or mitosis. The authors then validated the approach by
imaging changes in the surface exposure of a proto-cadherin, which was found to accumulate at the
surface of mitotic cells.
Prior to this study, little work was done to address the changes in the plasma membrane proteome
during cell cycle progression. Since this area has the potential to shed light on the changes in vesicle
trafficking and cortical organization that underlie changes in mitotic cell shape and that contribute to
changes in the dependence of cancer cells to growth and survival factor signaling when in mitosis,
the paper addresses an important area of cell biological research.
While the technical achievement is impressive, however, the data do not provide real insights into
functionally important changes in plasma membrane composition during cell cycle progression or
into changes in plasma membrane trafficking that have previously been linked to entry into mitosis.
For this reason, I think the paper would be more suited to a more specialized journal where the
methodological innovations will find an appreciative audience.
Comments
It is not clear why the authors permeabilized samples prior to streptavidin staining. "so the reagent
could access both surface-exposed and interior biotins." Doesn't this defeat the object of the study?
The text states "The immunoblots in Figure 2B used ultra agarose streptavidin beads when
compared with magnetic beads, we noticed a considerable decrease in cytoplasmic contaminants as
judged by the amount of actin in the elution (Figure 2C)." This is not clear.
Figure 2B-C
It is not clear why data are presented showing a poor purification prior to optimization of the
protocol.
Figure 2D
The text states: "525 of those were annotated as plasma membrane proteins, which corresponded to
the 17 % of the proteins identified in the search."
The authors should provide an objective measure to determine if this represents a good or bad yield.
Why isn't this in the Pie chart? What is the point of the graph in 2D? It doesn't provide the reader
with any meaningful data other than making clear the unexplained problem associated with amine
biotinylation of cytoplasmic proteins while "for membrane proteins (excluding mitochondrial and
nuclear membranes), the proteins isotopic ratio is more evenly distributed between carboxyl versus
amine biotinylation based sample preparation (Figure 2D, left, orange curve)."
If the caboxylate method is better why was the experiment repeated 7 times, 6 with amine labeling
and 1 with carboxylate?
Figure 4
The graph axes are not clear.
Moreover, the graph does not provide the reader with meaningful data.
Is it really necessary to dedicate two Figures to a GO term analysis?
"Proteins involved in cell projection appear to be more significantly enriched in
mitosis (Figure 4)". What does this mean?
Figure 5 does not do enough to show changes in the sub-cellular localization of PCDH7, its levels,
or the impact of these changes for protein function. Moreover, the focus on a single protein doesnt
do much to validate the systematic proteomic analysis undertaken.
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2013-85162
Figure 5C
It appears that not all cells in the Figure have GFP construct. Is this the case?
Figure 5D
What are the arrows pointing to? This Figure appears to show a GFP movie, but upon closer
examination appears to be a collage of images taken at different intensities. "Summarization of timelapse movies taken from U2OS cells expressing GFPPCDH7 and mcherry-tubulin. Images were
acquired every 20 minutes. Times are in minutes relative to the onset of mitosis." This is misleading,
especially as the 20' timepoint is overexposed. The implication is that GFP levels increase upon
entry into mitosis even when the protein is expressed from a heretologous promoter.
Referee #2:
Özlü et al. aimed to identify quantitative changes in the cell surface proteome between interphase
and mitosis. For the enrichment and the identification of cell surface proteins, they applied two
different labelling strategies, targeting amine groups and carboxyl groups in combination with
subsequent mass spectrometry-based analysis. For quantification they employed the SILAC
approach. Several cell surface proteins were found to be significantly enriched in either mitosis or
interphase, of which only one protein, PCDH7, was further validated. The authors claim to be the
first for using carboxyl labelling of cell surface proteins in combination with MS analysis.
Specific major concerns:
1. The authors present a complementary strategy for labeling cell surface proteins via previously
established EDC chemistry, but now in the context of living cells. The authors claim to label cells in
a buffer at pH 5-6 which typically leads to extensive cell death. I would like to see further control
experiments, including FACS, providing evidence for the viability of the cells during this harsh
treatment.
2. The list of 900 reported cell surface protein is not correct. Suppl. table 3 contains many duplicate
entries. The list contains only 534 unique proteins entries, which still is a reasonable coverage, but
the numbers mention in the text need to be corrected accordingly. The authors should further clarify
which peptides/ proteins were found with which technology (amine vs carboxy strategy).
3. There are several concerns regarding Table 1:
- The proteins in this table are referred to be significantly enriched on either state, but there are 6
protein listed with p-values >0.05. They are not significant.
- The ratio reported is very low for many proteins. What threshold for enrichment has been used?
Ratios below 2 (or 0.5) need to be justified.
- It is unclear what is meant by "Median Regulation" and how it differs from Ratio (Median SILAC
ratio).
- Also the numbers of significantly enriched proteins mention in the text need to be adjusted
accordingly.
4. There is no basic description in the Material and Method section about the mass spectrometric
setup used (LC gradient, technical replicates, type of MS instrument)
5. It is missleading, what the authors want to state regarding PCDH1. PCDH1 is also a
protocadherin, like PCDH7, which was validated. But even though is has the highest ratio, the
assigned p-value is borderline. So the authors decided, not to follow up on PCDH1. Nevertheless,
they hypothesize in the discussion that both protocadherin are expressed in a cell cycle dependent
manner at the cell surface and function together to down-regulate adhesion during mitosis. So, if the
authors hypothesize like that, they should also show immunostainings against PCDH1. Or on the
other hand, omit this conclusion.
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2013-85162
6. It looks like the authors only used oxidized Methionine and Carbomidomethylation, but forgot to
search for (or to mention that they did search for) deamidated peptides and the NHS residual tag on
carboxy groups and lysines. Please correct and/or comment.
7. References: It is quite interesting to note that the authors forgot/omitted the following references
which should, actually must, be included in this manuscript in form of at least citations, if not
discussed further as altenrative ways to explore and get coverage of the cell surface proteome.
Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans.
Ramya TN, Weerapana E, Cravatt BF, Paulson JC. Glycobiology. 2013 Feb;23(2):211-21. doi:
10.1093/glycob/cws144. Epub 2012 Oct 15.also Wollscheid et al. 2009 should be referenced for
carbohydrate labelling.
High-efficiency labeling of sialylated glycoproteins on living cells.
Zeng Y, Ramya TN, Dirksen A, Dawson PE, Paulson JC. Nat Methods. 2009 Mar;6(3):207-9. doi:
10.1038/nmeth.1305. Epub 2009 Feb 22.
PMID: 19234450
Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.
Wollscheid B, Bausch-Fluck D, Henderson C, O'Brien R, Bibel M, Schiess R, Aebersold R, Watts
JD. Nat Biotechnol. 2009 Apr;27(4):378-86. doi: 10.1038/nbt.1532. Epub 2009 Apr 6
Minor concerns:
1. It would be important to provide the reader with a visualization of the overlap (and differences)
between the cell surface proteins identified by amine labelling and carboxyl labelling, eg. in a venn
diagram.
2. The authors tried to find an optimal workflow and tested different affinity beads. Nevertheless, it
is unclear what was used in the final experiments. If they used a different setup for the different
replicates, it could be indicated in Suppl. table 1.
3. In the carboxyl labelling workflow, the cells were incubated with EDS and Sulfo-NHS in pH5.
The authors should assess the viability of the cells after this 15min incubation in order to ensure
exclusive (or close to exclusive) cell surface labelling.
4. Fig 4: The authors should indicate how many proteins were assigned into each category.
Otherwise the table can be partly misleading. In addition, the comparison should be carried out
against all PM assigned proteins by GO, rather than the whole IPI database.
Corrections:
1. Introduction:
The authors should explain the following sentence: "..long sample processing could facilitate
biochemical equilibration between two two-biochemical states." Do you mean that heavy labelled
proteins exchange their amino acids with the light proteins? It should be explained what kind of
biochemical equilibration could take part.
2. Results:
Wrong figure reference: "During this transition PCDH7 localizes to the retracted cell margin (Figure
5D, 20min.,)" Instead of 0 min!
Referee #3 :
The manuscript describes the comparative analysis of interphase versus mitotic plasma membrane
proteins by quantitative proteomics. This is a first as far as I am aware of. Surface proteins were
either labelled by a commercial NHS-activated reagent or by a self-synthesised reagent targeting
carboxy-groups. Both reagents should not permeate membranes and hence selectively label cell
surface proteins. Others have shown in the past that the labelling is indeed not as selective regarding
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2013-85162
cell surface as one should think and wishes for. Presumably this was also the case here and the
analysis was bioinformatically focused by using GO as a filter for previously annotated membrane
proteins. Overall I found the study enriching and it is likely to form the basis of or inspire future
work. I do have a number of concerns but feel that they could be addressed in a revised version of
the current manuscript.
The manuscript attracts three lines of criticism at this point. Firstly, the carboxyl-labelling is
technically not compared well to amine-labelling. Secondly, the full data is missing from the
supplementary tables. Thirdly, one might want to see an siRNA knock down of PCDH7 to test for a
possible phenotype in mitosis.
"While carboxyl group labeling with biotin provided more selective cell surface enrichment,
labeling both amine and carboxyl groups cast a wider net." The first part of this statement from the
abstract is not fully qualified based on a single experiment (consider the full and sound statistical
analysis of the significant changes in comparison). The second part really needs some Venn
diagrams. Similarly, Fig 2D is lacking numbers on the pie chart and leaves open what is actually
plotted in the graph (the overlap of what experiments?). "Although carboxylate biotinylation was
more cell-surface selective, the amine biotinlyation was advantageuos in that commercial reagents
and fewer processing steps were used." The argument is taken. Yet, to really say something about
the success of the authors own new strategy one would whish a second data set for comparison.
The authors focus the results of their analysis on those proteins with prior annotation as membrane
proteins according to GO. This limits the value of the resource offered by the authors more
significantly than the authors may imagine. It also conflicts with the established guidelines of the
proteomics community. It is acceptable that the quantitative analysis is focussed onto a filtered set
of identified and quantified proteins. However, all identified and quantified proteins need to be
listed and the data needs to be provided for all experiments. Obviously the list exists in the hands of
the authors as it formed the basis for their statistical analysis. Focussing on what GO "thinks" is in
membranes limits the discovery of truly novel factors.
Minor points:
"generates a mitotic arrest that is more similar to normal metaphase than anti-microtubule drugs."
Should really be changed to "...in this respect is more similar..."
"...and not blocking cleavage sites for LysC and trypsin" Sounds like a red herring to me. To my
knowledge there is no report that finds this to be a problem for NHS-activated reagents and also the
authors own success using NHS-biotin really speaks against this being of any concern.
"Activation of carboxylate groups has often been used for protein and peptide modification, but has
not before been used for labeling live mammalian cells to our knowledge." Could the activated
carboxyl-groups react with primary amines in the protein? This might explain why excessive reagent
amounts are needed, as the authors state at some point.
Is the elution from the beads really needed?
"(Stein & Berestecky, 1974). Johnsen et al (1975)," Could there be an error in reference formatting
here?
"...and interphase we then confirmed..." punctuation missing.
Fig 2D has a left/right confusion when being mentioned in the text. Why is it actually not called as
D and E? The "yellow" looks like brown to me.
"Therefore amine labeling was used for most experiments, while carboxylate labeling for
confirmation and to extend of our dataset." "of" is redundant
All three supplementary tables title "supplementary table 1".
"The width of the distribution is probably reduced because normalization minimized the variability."
Between experiments?
"However, for PCDH1...Therfore...,but..." is confusingly phrased and could be said clearer.
"mitosis normally-ie without" space missing.
"we utilized the (BAC transgenomics pipeline - Hela kyoto cell lines which stably express GFP
tagged proteins are under control of their own promoter)" use of parentheses unclear.
GO needs to be capitalized.
Re-submission
© European Molecular Biology Organization
03 February 2014
5
Thank you for sending our manuscript out for review last year. We appreciate the
comments that we received from the reviewers and have worked hard over the last year
to address these comments. The major criticism we received from reviewers was that
the study lacked functional analysis. In this version of the manuscript we addressed this
aspect. We feel this has greatly improved the manuscript and ask that you reconsider
our study for publication in EMBO. The change in the composition of the cell membrane
during the cell cycle has been understudied and is of general interest to the wide
audience of the EMBO journal. Please find below our point by point comments and
responses to the reviews.
Thank you in advance for your time.
Sincerely
Nurhan Ozlu and Judith Steen
Referee #1 :
In this paper, Alper et al. describe a new method by which to assess differences in the
protein composition of the mitotic and interphase plasma membrane. This required the
team to develop a protocol in which isotope labeling together with amine and carboxylate
chemistry are used to differentially mark and purify biotinylated surface proteins in
mitotic and interphase cells.
In this way the authors were then able to use MS to identify a long list of proteins that
preferentially accumulate on the cell surface in interphase or mitosis. The authors then
validated the approach by imaging changes in the surface exposure of a proto-cadherin,
which was found to accumulate at the surface of mitotic cells.
Prior to this study, little work was done to address the changes in the plasma membrane
proteome during cell cycle progression. Since this area has the potential to shed light on
the changes in vesicle trafficking and cortical organization that underlie changes in
mitotic cell shape and that contribute to changes in the dependence of cancer cells to
growth and survival factor signaling when in mitosis, the paper addresses an important
area of cell biological research.
While the technical achievement is impressive, however, the data do not provide real
insights into functionally important changes in plasma membrane composition during cell
cycle progression or into changes in plasma membrane trafficking that have previously
been linked to entry into mitosis. For this reason, I think the paper would be more suited
to a more specialized journal where the methodological innovations will find an
appreciative audience.
We thank the reviewer for their general comments and for recognizing the novelty of this
study. We address the major criticism that that while the study is a methodological
innovation no functional insights were derived for the cell cycle importance of the
changes in the plasma membrane. We spent the last year studying focusing on
the two highest scoring mitosis-selective cell surface proteins: PCDH1 and PCDH7. We
analyzed their function in mitosis and show that these proteins are required for the
increase in rounding pressure at the onset of mitosis. The knockdown of PCDH1 and 7
caused a decrease in mitotic rounding pressure and we further showed that PCDH7
can be used as a proliferative maker in intact cells. To our knowledge this type of
regulation is unprecedented and has not been reported in the literature, and it adds
significantly to our knowledge of the little-studied vertebrate protocadherins.
Comments
It is not clear why the authors permeabilized samples prior to streptavidin staining. "so
the reagent could access both surface-exposed and interior biotins." Doesn't this defeat
the object of the study?
We apologize for not making this clear in the previous manuscript. Permeabilization was
done to test the biotinylation proceedure in cells with microscopy but was not used for
cell surface preparation procedure. The purpose of permeabilization was to be able to
judge the amount of cell-exposed biotin versus interior biotin and the efficiency of the
biotin labeling procedure. Importantly. our data shows that despite the presence of biotin
in the cytoplasm of the cells after treating live cells with non-permeable amine reactive
biotinylation reagent, Sulfo-NHS-SS-Biotin and fixation, even after permeabilization,
fluorescent streptavidin was found to intensely stain the cell surface due to high
concentration of biotin on the cell surface. We also performed the staining without
permeabilization. To clarify, all membrane preparations for the proteomics experiments
were performed without the permeabilization step.
The text states "The immunoblots in Figure 2B used ultra agarose streptavidin beads
when compared with magnetic beads, we noticed a considerable decrease in
cytoplasmic contaminants as judged by the amount of actin in the elution (Figure 2C)."
This is not clear.
Figure 2B-C
It is not clear why data are presented showing a poor purification prior to optimization of
the protocol.
This immunoblot was intended to show the our optimization procedure and the
improvents that we achieved, however based on reviewer's comment the presentation of
the un-optimized data is unnecessary. As such this figure has been modified and only
shows immunoblots of an optimized protocol.
Figure 2D
The text states: "525 of those were annotated as plasma membrane proteins, which
corresponded to the 17 % of the proteins identified in the search."
The authors should provide an objective measure to determine if this represents a good
or bad yield. Why isn't this in the Pie chart? What is the point of the graph in 2D? It
doesn't provide the reader with any meaningful data other than making clear the
unexplained problem associated with amine biotinylation of cytoplasmic proteins while
"for membrane proteins (excluding mitochondrial and nuclear membranes), the proteins
isotopic ratio is more evenly distributed between carboxyl versus amine biotinylation
based sample preparation (Figure 2D, left, orange curve)."
If the caboxylate method is better why was the experiment repeated 7 times, 6 with
amine labeling and 1 with carboxylate?
We agree that the carboxyl labeling methodology were not represented to a similar
extend to amine labeling. Based on reviewer's comment, we decided to focus on the
amine labeling; detailed characterization of carboxyl labeling will be reported elsewhere.
Figure 4
The graph axes are not clear.
Moreover, the graph does not provide the reader with meaningful data.
Is it really necessary to dedicate two Figures to a GO term analysis?
"Proteins involved in cell projection appear to be more significantly enriched in
mitosis (Figure 4)". What does this mean?
We agree that GO analysis by itself is not very informative therefore; this figure was
removed from the result section.
Figure 5 does not do enough to show changes in the sub-cellular localization of PCDH7,
its levels, or the impact of these changes for protein function. Moreover, the focus on a
single protein doesnt do much to validate the systematic proteomic analysis undertaken.
In the revised manuscript, we greatly extended this aspect of the study in response to
the reviewer. In the previous submission we performed immunostaining of endogenous
protein, expressed PCDH7::GFP under its own promoter using Bacterial artificial
chromosome (BAC) transgenomics, and performed live imaging studies of the the
transiently expressed PCDH7::GFP fusion gene, In the revised manuscript we did many
additional experiments: We confirmed the subcellular localization result using western
blotting (see Figure 2B) and FACS analysis (see Figure 6). Further, we quantified the
PCDH7 signal in immunostained and PCDH7::GFP expressing mitotic and interphase
cells (Figure 3B and Figure 4B) to quantitatively show the enrichment of PCDH7 at the
cell surface during mitosis. We provided more insight into the subcellular localization of
PCDH7 by examining subcellular localization of PCDH7 when it not on the cell surface.
In interphase cells, PCDH7 appeared to localize to endomembranes that were close to
the ER marker calnexin (Figure 4C) (co-fractionated with ER by density gradient (Figure
4D). To better understand the biochemistry of this localization to the endomembranes
we perfomed domain analysis of PDCH7. Our data showed that the relatively short
cytoplasmic domain of the PCDH7 was required for retention in this compartment –
PCDH7 lacking this domain was mislocalized to the cell surface in interphase (Figure
4A-B). Our statistical analysis of the of the amine biotinylation labeling proteomic data
showed that PCDH1 was selectively localized to the mitotic cell surface. Mitosis
selective cell surface exposure of PCDH1 was also confirmed by western blotting
analysis (Figure 2C). To better understand protein function, of PDCH1 and PDCH7 we
performed knock-down experiments. Using AFM microscopy, we showed that a
knockdown of PCDH1 and 7 caused a decrease in mitotic rounding pressure (Figure 5).
Futhermore, using flow cytometry we found that live cells incubated with anti-PCDH7
antibody and subsequently fixed can be enriched for the mitotic population based on
their PCDH7 expression (Figure 6).
Prior to this work, not much is known about PCDH1 and PCDH7, however using
advanced methodologies we provided novel information about these proteins and
proposed that the surface-exposed portion of PCDH7 can be used as imaging agents for
detecting mitotic cells in situ. Our analysis also confirmed known proteins that are
enriched on the cell surface in cell cycle dependent manner; TFRC is one of the most
interphase-enriched surface proteins. In the future we will focus on several other novel
candidates that were identified in this study.
Figure 5C
It appears that not all cells in the Figure have GFP construct. Is this the case?
That is correct it a poll of cells instead of a single colony, therefore not all cells express
bacterial artificial chromosome.
Figure 5D
What are the arrows pointing to?
Arrows are pointing to the mitotic cells surrounded by the interphase cells to highlight the
mitotic ones. An explanation about the arrows was included in the figure legend. To
make the arrows less confusing to the reader, in the revised version arrows are moved
to the merged picture from the only PCDH7 staining picture.
This Figure appears to show a GFP movie, but upon closer examination appears to be a
collage of images taken at different intensities. "Summarization of time-lapse movies
taken from U2OS cells expressing GFPPCDH7 and mcherry-tubulin. Images were
acquired every 20 minutes. Times are in minutes relative to the onset of mitosis." This is
misleading, especially as the 20' timepoint is overexposed. The implication is that GFP
levels increase upon entry into mitosis even when the protein is expressed from a
heterologous promoter.
In all movies provided in the supplementary (and snapshot images shown in Figure 5D,
in the revised version Figure 3E) none of the timepoints had different exposure time. The
experiment used an overnight movie set-up to take images every 20 minutes at a fixed
exposure time. This movie is extremely informative and shows the dynamic cortex
localization of PCDH7 at the onset of mitosis. To confirm the PCDH7's cortex localization
more quantitatively, in the revised version we quantified the PCDH7 signal on the cell
surface of both immuno-stained cells (endogenous protein) and transiently PCDH7 cells
(under heterologous promoter) (Figure 3C and Figure 4B). In both cases the
surface/interior ratios showed that wild-type PCDH7 was highly surface enriched during
mitosis.
Referee #2 :
Özlü et al. aimed to identify quantitative changes in the cell surface proteome between
interphase and mitosis. For the enrichment and the identification of cell surface proteins,
they applied two different labelling strategies, targeting amine groups and carboxyl
groups in combination with subsequent mass spectrometry-based analysis. For
quantification they employed the SILAC approach. Several cell surface proteins were
found to be significantly enriched in either mitosis or interphase, of which only one
protein, PCDH7, was further validated. The authors claim to be the first for using
carboxyl labelling of cell surface proteins in combination with MS analysis.
Specific major concerns:
1. The authors present a complementary strategy for labeling cell surface proteins via
previously established EDC chemistry, but now in the context of living cells. The authors
claim to label cells in a buffer at pH 5-6 which typically leads to extensive cell death. I
would like to see further control experiments, including FACS, providing evidence for the
viability of the cells during this harsh treatment.
We appreciate the criticisms that the carboxyl labeling methodology was not repeated or
represented to the same extent as the amine labeling. Based on reviewer's comment,
authors decided to focus on the amine labeling; detailed characterization of carboxyl
labeling will be reported elsewhere.
2. The list of 900 reported cell surface protein is not correct. Suppl. table 3 contains
many duplicate entries. The list contains only 534 unique proteins entries, which still is a
reasonable coverage, but the numbers mention in the text need to be corrected
accordingly. The authors should further clarify which peptides/ proteins were found with
which technology (amine vs carboxy strategy).
The duplicate entries were due to the redundancy in the IPI database; we recounted the
unique protein entries with the NCBI gene names in this version.
3. There are several concerns regarding Table 1:
- The proteins in this table are referred to be significantly enriched on either state, but
there are 6 protein listed with p-values >0.05. They are not significant.
Table 1 listed examples of mitosis- and interphase-enriched and unchanged cell surface
proteins. Therefore p-values >0.05 are examples for the unchanged proteins. To avoid
confusion, in the title, it was stated that in addition to the significantly enriched ones,
representative of unchanged proteins were also listed.
- The ratio reported is very low for many proteins. What threshold for enrichment has
been used? Ratios below 2 (or 0.5) need to be justified.
Proteins are listed as enriched when they have a multiply-corrected p-value of below
0.05. While in most cases, this coincides with having a normalized SILAC ratio of below
0.5 or above 2, this does not hold for all cases. In our analysis, 15 statistically significant
mitotic proteins had the median ratio above 2.5 fold (in the range of 2.5-8) and around
20 statistically significant interphase proteins had a median SILAC ratio of less than
0.45. Here, we rely on the p-value since it is the more objective criterion and less likely
to be perturbed by variation (Herrmann et al., 2013).
13
- It is unclear what is meant by "Median Regulation" and how it differs from Ratio
(Median SILAC ratio).
We apologize for the confusion; Median Regulation is the same thing as the normalized
Median SILAC ratio. We changed the median regulation with median (normalized) ratio.
- Also the numbers of significantly enriched proteins mention in the text need to be
adjusted accordingly.
4. There is no basic description in the Material and Method section about the mass
spectrometric setup used (LC gradient, technical replicates, type of MS instrument)
We apologize for this omission. In the Material Method section we included a section
explaining LC gradient, type of MS instruments and data handling. Please see "Materials
and Methods-Mass Spectrometry and Data Acquisition". In our experiments, we started
our experiments with 4 jumbo plate Hela cells (4*106), after cell surface isolation via
biotinylation, we digested the resulting peptides and separated into 24 fractions by
isoelectric focusing. Each fraction we preferred to run in the LC/MS using a long gradient
once (without a technical repeat) due to limitation in the material and instrumentation
time.
5. It is missleading, what the authors want to state regarding PCDH1. PCDH1 is also a
protocadherin, like PCDH7, which was validated. But even though is has the highest
ratio, the assigned p-value is borderline. So the authors decided, not to follow up on
PCDH1. Nevertheless, they hypothesize in the discussion that both protocadherin are
expressed in a cell cycle dependent manner at the cell surface and function together to
down-regulate adhesion during mitosis. So, if the authors hypothesize like that, they
should also show immunostainings against PCDH1. Or on the other hand, omit this
conclusion.
In the revised version the localization of PCDH1 in mitosis became clearer . When the
carboxyl labeling data were excluded and p-values re-calculated, PCDH1 was
significantly enriched at the mitotic membrane. We confirmed this using western blotting
(Available commercial Antibodies did not work for immunostaining). In addition
knockdown of PCDH1 using RNAi showed that PCDH1 is required for building rounding
pressure at the onset of mitosis.
6. It looks like the authors only used oxidized Methionine and Carbomidomethylation, but
forgot to search for (or to mention that they did search for) deamidated peptides and the
NHS residual tag on carboxy groups and lysines. Please correct and/or comment.
Variable modifications were kept minimal to lower the complexity of the Mascot Search.
Therefore Cysteine carbamidomethylation was used as a fixed modification, and
oxidation (M), N-acetyl (protein N-term) were set as variable modifications efficiency. We
did not include the residual tag on carboxyl groups as the tag probably causes trypsin
misscleavage given the low number of peptides identified with the tag in initial
experiments.
7. References: It is quite interesting to note that the authors forgot/omitted the following
references which should, actually must, be included in this manuscript in form of at least
citations, if not discussed further as altenrative ways to explore and get coverage of the
cell surface proteome.
We thank the reviewer for the input; following citations are added to the current version
of the manuscript.
Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans.
Ramya TN, Weerapana E, Cravatt BF, Paulson JC.
Glycobiology. 2013 Feb;23(2):211-21. doi: 10.1093/glycob/cws144. Epub 2012 Oct
15.also Wollscheid et al. 2009 should be referenced for carbohydrate labelling.
High-efficiency labeling of sialylated glycoproteins on living cells.
Zeng Y, Ramya TN, Dirksen A, Dawson PE, Paulson JC.
Nat Methods. 2009 Mar;6(3):207-9. doi: 10.1038/nmeth.1305. Epub 2009 Feb 22.
PMID: 19234450
Mass-spectrometric identification and relative quantification of N-linked cell surface
glycoproteins.
Wollscheid B, Bausch-Fluck D, Henderson C, O'Brien R, Bibel M, Schiess R, Aebersold
R, Watts JD.
Nat Biotechnol. 2009 Apr;27(4):378-86. doi: 10.1038/nbt.1532. Epub 2009 Apr 6
Minor concerns:
"We addressed all the topics of "minor concern" listed below in the revised version."
1. It would be important to provide the reader with a visualization of the overlap (and
differences) between the cell surface proteins identified by amine labelling and carboxyl
labelling, eg. in a venn diagram.
2. The authors tried to find an optimal workflow and tested different affinity beads.
Nevertheless, it is unclear what was used in the final experiments. If they used a
different setup for the different replicates, it could be indicated in Suppl. table 1.
3. In the carboxyl labelling workflow, the cells were incubated with EDS and Sulfo-NHS
in pH5. The authors should assess the viability of the cells after this 15min incubation in
order to ensure exclusive (or close to exclusive) cell surface labelling.
4. Fig 4: The authors should indicate how many proteins were assigned into each
category. Otherwise the table can be partly misleading. In addition, the comparison
should be carried out against all PM assigned proteins by GO, rather than the whole IPI
database.
Corrections:
1. Introduction:
The authors should explain the following sentence: "..long sample processing could
facilitate biochemical equilibration between two two-biochemical states." Do you mean
that heavy labelled proteins exchange their amino acids with the light proteins? It should
be explained what kind of biochemical equilibration could take part.
2. Results:
Wrong figure reference: "During this transition PCDH7 localizes to the retracted cell
margin (Figure 5D, 20min.,)" Instead of 0 min!
Referee #3 :
The manuscript describes the comparative analysis of interphase versus mitotic plasma
membrane proteins by quantitative proteomics. This is a first as far as I am aware of.
Surface proteins were either labelled by a commercial NHS-activated reagent or by a
self-synthesised reagent targeting carboxy-groups. Both reagents should not permeate
membranes and hence selectively label cell surface proteins. Others have shown in the
past that the labelling is indeed not as selective regarding cell surface as one should
think and wishes for. Presumably this was also the case here and the analysis was
bioinformatically focused by using GO as a filter for previously annotated membrane
proteins. Overall I found the study enriching and it is likely to form the basis of or inspire
future work. I do have a number of concerns but feel that they could be addressed in a
revised version of the current manuscript.
The manuscript attracts three lines of criticism at this point.
Firstly, the carboxyl-labelling is technically not compared well to amine-labelling.
We agree with this criticism. Based on all reviewers' comment, we decided to focus on
the amine labeling; detailed characterization of carboxyl labeling will be reported
elsewhere.
Secondly, the full data is missing from the supplementary tables.
This is a valid point, in the revised version; we provided all the data together with their
mitosis/interphase ratio. Please see Supplementary Table 4.
Thirdly, one might want to see an siRNA knock down of PCDH7 to test for a possible
phenotype in mitosis.
We thank the reviewers for this suggestion and did this experiment, RNAi knockdown of
PCDH7 showed that PCDH7 is required for building rounding pressure at the onset of
mitosis. We greatly extended our work to understand the molecular mechanism and
function of cell cycle dependent cell surface exposure of PCDH7 in the revised
manuscript.
"While carboxyl group labeling with biotin provided more selective cell surface
enrichment, labeling both amine and carboxyl groups cast a wider net." The first part of
this statement from the abstract is not fully qualified based on a single experiment
(consider the full and sound statistical analysis of the significant changes in comparison).
The second part really needs some Venn diagrams. Similarly, Fig 2D is lacking numbers
on the pie chart and leaves open what is actually plotted in the graph (the overlap of
what experiments?). "Although carboxylate biotinylation was more cell-surface selective,
the amine biotinlyation was advantageuos in that commercial reagents and fewer
processing steps were used." The argument is taken. Yet, to really say something about
the success of the authors own new strategy one would whish a second data set for
comparison.
The carboxyl labeling was excluded in the revised version of the manuscript. This
method will be published as a separate analysis elsewhere.
The authors focus the results of their analysis on those proteins with prior annotation as
membrane proteins according to GO. This limits the value of the resource offered by the
authors more significantly than the authors may imagine. It also conflicts with the
established guidelines of the proteomics community. It is acceptable that the quantitative
analysis is focused onto a filtered set of identified and quantified proteins. However, all
identified and quantified proteins need to be listed and the data needs to be provided for
all experiments. Obviously the list exists in the hands of the authors as it formed the
basis for their statistical analysis. Focussing on what GO "thinks" is in membranes limits
the discovery of truly novel factors.
This is a valid point in the revised version, authors provided all identified and quantified
proteins from all repeats together with their mitosis/interphase ratio (Supplementary
Table4). In this study we only focused on the cell surface proteins based on their GO
annotation. However all data is available as a resource to the public.
Minor points:
All the minor points are addressed in the current version.
"generates a mitotic arrest that is more similar to normal metaphase than antimicrotubule drugs." Should really be changed to "...in this respect is more similar..."
The sentence is changed as: A kinesin-5 inhibitor was chosen for mitotic arrest because
it leaves microtubule dynamics intact; in this respect it generates a mitotic arrest that is
more similar to normal metaphase than anti-microtubule drugs.
"...and not blocking cleavage sites for LysC and trypsin" Sounds like a red herring to me.
To my knowledge there is no report that finds this to be a problem for NHS-activated
reagents and also the authors own success using NHS-biotin really speaks against this
being of any concern.
This sentence has been removed.
"Activation of carboxylate groups has often been used for protein and peptide
modification, but has not before been used for labeling live mammalian cells to our
knowledge." Could the activated carboxyl-groups react with primary amines in the
protein? This might explain why excessive reagent amounts are needed, as the authors
state at some point.
Carboxyl labeling method has been removed from the revised manuscript.
Is the elution from the beads really needed?
Elution from the beads is needed for western blotting. For mass spectrometry analysis,
protein digestion can be performed on the beads without elution whereas for western
blotting eluting with DTT is absolutely necessary. Regardless we did the elution from the
beads both for western blotting and MS analysis.
"(Stein & Berestecky, 1974). Johnsen et al (1975)," Could there be an error in reference
formatting here?
Thank you for pointing this mistake, we corrected it in the revised version.
"...and interphase we then confirmed..." punctuation missing.
Fig 2D has a left/right confusion when being mentioned in the text. Why is it actually not
called as D and E? The "yellow" looks like brown to me.
"Therefore amine labeling was used for most experiments, while carboxylate labeling for
confirmation and to extend of our dataset." "of" is redundant
These sentences were removed in the revised version.
All three supplementary tables title "supplementary table 1".
This is corrected in the revised version.
"The width of the distribution is probably reduced because normalization minimized the
variability." Between experiments?
"However, for PCDH1...Therfore...,but..." is confusingly phrased and could be said
clearer.
"mitosis normally-ie without" space missing.
These sentences were removed in the revised version.
"we utilized the (BAC transgenomics pipeline - Hela kyoto cell lines which stably express
GFP tagged proteins are under control of their own promoter)" use of parentheses
unclear.
Parentheses are removed.
GO needs to be capitalized.
The EMBO Journal Peer Review Process File - EMBO-2013-85162
2nd Editorial Decision
14 April 2014
Thank you for your patience with the assessment of your resubmitted Resource Article on cell
surface proteome comparison. Referees 1 and 3 have now looked at it once more, and we have
discussed their input at length within our editorial team. As you will see, referee 1 - while
acknowledging the improvements and extension of the cell biological work - is still not convinced
that this part of the analysis is sufficiently conclusive, both regarding the understanding of
protocadherin function and the mechanism of its regulation. Referee 3 considers the manuscript
generally improved and now in principle suitable for publication as a Resource Article, but still
retains a number of reservations regarding the description, presentation and interpretation of
especially the proteomics data.
Given the potential interest of your added follow-up work, and the principle importance of the
described cell surface proteomics data sets, we concluded that we may be able to consider this
manuscript further through an exceptional additional round of revision, but only in the case you
should be prepared and able to address the following essential issues:
- it will be absolutely required at this stage to fully address referee 3's comments on the presentation
and description of the proteomics data, especially since similar concerns had already been initially
raised by this reviewer.
- regarding PCDH7 analysis, a number of specific aspects need to be clarified, such as determining
the percentage of phospho-H3-positive cells that are also PCDH7-positive, and
clarifying/strengthening PCDH7 localization aspects (referee 1) and levels/localization of
endogenous protein (referee 3). On the other hand, we feel that understanding the mechanistic basis
for PCDH7 (re-)localization may be beyond the scope of the present work.
- with regard to PCDH knockdown phenotypes, RNAi rescue experiments (as requested by referee
1) will be essential to support the functional conclusions. Moreover, any additional data on how the
rounding pressure phenotypes may affect mitotic events/progression would clearly be helpful in
order to strengthen the functional relevance of the observed role.
- finally, I noticed that the data quality in the figures, especially of blots/gels/autoradipgraphs, is
currently too low for publication or even for proper assessment of these data and their relation to the
original data. Upon resubmission, please therefore provide all source data files for these panels. We
would ask for a single PDF/JPG/GIF file per figure comprising the original, uncropped and
unprocessed scans of all gel/blot panels used in the respective figures, to be uploaded selecting
"Figure Source Data" as object type. These should be labelled with the appropriate figure/panel
number, and should have molecular weight markers; further annotation would clearly be useful but
is not essential. Furthermore, please reassemble all figures with data of sufficient resolution,
avoiding over-adjustments of contrast and brightness so as to not obscure the original data, and
clearly indicating by dividing lines and explanations in the figure legends all cases were nonadjacent lanes have been placed next to each other.
In case you should decide to resubmit a re-revised manuscript (using the link provided below),
please bear in mind that this will be the final round of revision we can consider here, and that all the
above points (as well as the various more specific issues raised by the reviewers) will need to be
decisively clarified and addressed. Should you have any questions/comments with regard to this
decision, the reports and the revision requirements, please do not hesitate to get back to me.
Referee #1:
The revised paper by Ozlu and colleagues is much improved. The author's have ironed out many of
the problems with the presentation of the biochemistry - mostly through omission of confusing data.
In their revised manuscript they have done much more to follow up work on the cell biology of hits
identified in the Mass spect analysis. They have focused their analysis on two proto-Cadherins that
appear to be trafficked to the cell surface during mitosis. They provide evidence that these
protocadherins provide useful markers of mitosis (what proportion of pH3 cells are positive for
PCDH7?), but provide little data to shed light on the function of these proteins or, more importantly
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given the subject of the paper, the mechanism by which they become concentrated on the surface of
mitotic cells.
The text suggests that PCDH7 becomes localized to the "cell cortex and nuclear membrane" and to
the "retracted cell margin" in cells entering mitosis. This is not at all clear from the data. It is not at
all clear what the arrows in 3E are supposed to reveal, and identify of the aggregates in 3E and 4A
has not been explained.
The authors also point to PCDH7 being in the ER. They state the it is "partially co-localized" with
calnexin. Again, this isn't clear in the data. In fact there is very little overlap, and the biochemistry
relies only on the isolation of two fractions, cytoplasm and ER. Further, the change in localisation
induced by a truncation of the tail leads does not remove the mitotic surface accumulation, leaving
the mechanism by which this pattern of localization is achieved unexplained.
The authors state that they were unable to identify a change in mitotic cell shape changes or mitotic
progression following gene silencing. While the authors appear to have identified a difference in the
rounding force under a cantilever following proto-cadherin RNAi, it is not clear what this would
correspond to - a change in ER organisation? Since the impact of all 3 RNAi treatments
(PCDH1/7/both) appear the same, more must be done to verify that this is meaningful, i.e. BAC
rescue and alternative control experiments, before conclusions can be made. Moreover, this seems a
extremely elaborate way of identifying an unexplained phenotype.
In summary, the paper still fails to combine two types of analysis. The mass spect analysis is
impressive, but will interest a specialized audience. The cell biology is inconclusive and doesn't
address the important question of how protocadherins make it to the surface in mitosis, when many
other surface proteins do not
Referee #3:
The revised manuscript presents increased biological validation of the resource by following on two
identified proteins. Preliminary data on carboxyl-targeting capture reagent was removed. Removal
of the carboxyl-targeting capture reagent has improved the manuscript in clarity. These data were
clearly preliminary and added therefore little to the main point of the manuscript. PCDH7 is now
presented as a cell-surface marker of mitotic Hela and MCF7 cells. Furthermore, this establishes an
experimental workflow for the comparison of interphase and mitotic cells that may ultimately lead
to the analysis of primary cells. This points towards interesting avenues of cancer research that are
appearing more possible with this work coming out.
For clarity and transparency the proteomics data should be described in completion, a pie chart of all
data should be shown with the slice that the remainder of the manuscript focuses on (GO-based
membrane) being highlighted. Possibly even more effective, a Venn diagram could show in one
circle the proteins detected in this study and in another those in GO marked as membrane
associated; the overlap would then be the ones followed further here. The GO-based focusing does
not appear in the current figure 1 and therefore -and also because of non-consistent wordingremains misleading. Not even Supplementary Table 1 lists the actual number of identified and
quantified proteins. GO-based filtering is an essential step of the actually used (but not in the
presented) experimental procedure and must feature in the flow chart, Figure 1C. Figure 1C is
misleading as it suggests that all results of the proteomic analysis are discussed in the manuscript
and shown in Figure 2A while it is actually a filtered subset. Supplementary Table 4 should have a
column stating if a protein group was included or not in the analysis. The table also misses
information needed to reanalyze it and obtain the same results as the authors. Given the focus on
resource this is essential.
P4: "mitotic proteins" proteins enriched in mitosis. To name them mitotic proteins a function would
need to be demonstrated in mitosis. Same for "interphase proteins".
P5: Reduced proteomic detection of PCDH7 in interphase does not necessarily suggest localization
of this protein in an internal compartment in interphase. Proteomic analysis bases on the protein
being accessible to labeling. The methods section is not clear on whether the HeLa S3 cells were
grown in suspension or on plates.
Was intracellular PCDH7 only quantified for overexpressed GFP-labeled protein? What happens
when using fixed cells and antibodies against the native protein?
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P13: Figure 3A left/right presumably refers to Figure 2. "all proteins"? Is this really true, all proteins
identified in the five replica or only the subset obtained by GO-based filtering? The details given
should allow a reader to reconstruct the findings based on Supplementary Table 4!
Author response letter: "The sentence is changed as: A kinesin-5 inhibitor was chosen for mitotic
arrest because it leaves microtubule dynamics intact; in this respect it generates a mitotic arrest that
is more similar to normal metaphase than anti-microtubule drugs." I cannot confirm this.
1st Revision - authors' response
25 August 2014
Reviewer #1:
The revised paper by Ozlu and colleagues is much improved. The author's have ironed out many
of the problems with the presentation of the biochemistry - mostly through omission of confusing
data.
In their revised manuscript they have done much more to follow up work on the cell biology of
hits identified in the Mass spect analysis. They have focused their analysis on two protoCadherins that appear to be trafficked to the cell surface during mitosis. They provide evidence
that these protocadherins provide useful markers of mitosis (what proportion of pH3 cells are
positive for PCDH7?), but provide little data to shed light on the function of these proteins or,
more importantly given the subject of the paper, the mechanism by which they become
concentrated on the surface of mitotic cells.
The text suggests that PCDH7 becomes localized to the "cell cortex and nuclear membrane" and
to the "retracted cell margin" in cells entering mitosis. This is not at all clear from the data. It is
not at all clear what the arrows in 3E are supposed to reveal, and identify of the aggregates in 3E
and 4A has not been explained.
The snap-shot images from the movies of the PCDH7::GFP expressing cell did not adequately
reflect the dynamics of PCDH7 localization. In the revised manuscript we now provide two movies
but do not try to summarize the movies with few frozen images. The dynamic localization of
PCDH7 is obvious in the movies. We revised the text as below:
To gain more insight into cell cycle dynamics of PDCH7, we performed live imaging studies in
transiently transfected U2OS cells. In interphase cells, the PCDH7 signal showed diffuse
localization in the cytoplasm. As the cell progressed into prometaphase and become rounded and
less adhesive, PCDH7 localized to the retraction fibers around the cortex and during metaphase
PCDH7 appeared to localize to the cell cortex . In the post mitotic daughter cells, PCDH 7 mostly
localized to the cell-cell contact margin and not to the rest of the cortex. See videos 1 and 2
(Supplementary Data).
We agree that aggregated protein is always a concern in over-expression situations. We observed
PCDH7 aggregates only after the transient transfection but not in the endogenous staining or
PCDH7::GFP BAC transgenomic cells. Therefore, aggregates are most likely an artifact of
GFP::PCDH7 over-expression. To address this concern we repeated the experiments with
endogenous PCDH7 and PCDH7::GFP BAC transgenomics expressing cells where possible.
The authors also point to PCDH7 being in the ER. They state the it is "partially co-localized"
with calnexin. Again, this isn't clear in the data. In fact there is very little overlap, and the
biochemistry relies only on the isolation of two fractions, cytoplasm and ER.
We agree that the co-localization data, and in general the evidence for ER retention of PCDH7 in
interphase, were weak in the previous submission. To address this important point raised by the
reviewer, we took better images of PCDH localization in interphase, and repeated the colocalization experiment with a new ER marker, KDEL, These data are provided in Figure 4C and
Supplementary Figure 1. Co-localization between PCDH7 and the ER markers in interphase cell is
much more obvious (see Figure 4C and suplementary Figure 1). , PCDH7 does not completely co-
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localize with the ER markers, and we are careful to say in the text that we cannot strongly conclude
that it is retained in the ER in interphase. It could be in some other compartment that is proximal to
the ER and has a similar density. However, we feel that the data allow us to propose ER retention in
interphase as a reasonable working model for cell cycle regulation of PCDH7 surface expression.
The reviewer made a reasonable point in criticizing the biochemical fractionation data in the
previous manuscript, which we acknowledge was preliminary. To address this criticism we repeated
and improved the fractionation experiment, and extended our data to probe markers for cytoplasm,
mitochondria, and ER. Each fraction of the density gradients was immunoblotted for an endoplasmic
reticulum protein, KDEL, a cytoplasmic protein; actin, a mitochondrial protein; cox-2. Both
PCDH7, PCDH1 were enriched in the ER fraction and were not detectable in the cytoplasmic or
mitochondrial fractions. We realize this is still an incomplete story, for example the ER fraction is
surely contaminated with other light membranes. However, we argue a deeper fractionations study
would go beyond the scope of this resource submission.
Further, the change in localization induced by a truncation of the tail leads does not remove the
mitotic surface accumulation, leaving the mechanism by which this pattern of localization is
achieved unexplained.
We agree that we have not solved the mechanism by which the cell surface exposure of PCDH1 and
7 is cell cycle regulated. We have made progress on this problem, and developed the beginnings of a
mechanistic hypothesis, where the cytoplasmic domain specifies ER retention in interphase cells,
but not in dividing cells. We feel the reviewer is asking too much, especially for a paper that was
submitted as a resource, and not as a cell biology mechanisms paper. We would remind the reviewer
that PCDH1 and 7 are the first examples of any protein that is selectively exposed on the surface of
dividing cells, and that our paper provides the community with the first ever, carefully documented,
list of proteins whose surface exposure is cell cycle regulated. We would like to solve the problem
of how PCDH7 exposure is cell cycle regulated, but we feel this problem should be further tackled
in follow-up studies, not in this paper.
The authors state that they were unable to identify a change in mitotic cell shape changes or
mitotic progression following gene silencing. While the authors appear to have identified a
difference in the rounding force under a cantilever following proto-cadherin RNAi, it is not clear
what this would correspond to - a change in ER organisation? Since the impact of all 3 RNAi
treatments (PCDH1/7/both) appear the same, more must be done to verify that this is meaningful,
i.e. BAC rescue and alternative control experiments, before conclusions can be made. Moreover,
this seems a extremely elaborate way of identifying an unexplained phenotype.
We have made multiple improvements in the in revised version to make the biologic analysis as
impressive as the mass spec. analysis, and to strengthen the functional analysis of protocadherins. A
BAC rescue experiment was done to shown that RNAi resistant protocadherin can rescue the
rounding force phenotype. Because no mouse BAC clone was available for PCDH7 but only
PCDH1 mouse BAC clone was available, the experiment could not be repeated for PCDH7.
Additionally to address the relevance of protocadherin function and to provide additional data on
how the rounding pressure phenotypes may affect mitotic events we carefully measured the speed of
cell rounding, we observed a slight decrease in decrease in cell rounding in PCDH1 RNAi
(Supplementary Figure 2A). Although this affect may seem subtle, it is significant for the reasons
mentioned above. In addition over-expression of the ectodomain of PCDH7 induced cell rounding in
interphase, and loss of focal adhesions suggesting an anti-adhesive function of the ectodomain
(Supplementary Figure 2B). All data together provide the first large-scale of cell surface proteome
between interphase and mitosis and it adds significantly to our knowledge of the relatively littlestudied vertebrate protocadherins.
In summary, the paper still fails to combine two types of analysis. The mass spect analysis is
impressive, but will interest a specialized audience. The cell biology is inconclusive and doesn't
address the important question of how protocadherins make it to the surface in mitosis, when
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many other surface proteins do not
We strongly context the reviewer’s opinion that our proteomic analysis will only be of interest to a
specialist audience. It will be of interest to groups interested in cell cycle regulation of endo- and
exocytosis, to groups interested in mitotic rounding which is currently a hot topic, and, we hope, to
students of tissue biology more broadly who would like clues as to how cell division impacts cellcell communication and adhesion. Groups interested in these topics have requested access to our
data pre-publication, which is one reason we chose to submit as a resource article. We have strived
to make the data accessible to this broad audience, and in the discussion have highlighted pathways
and proteins that we think are worthy of further study in light of the new information provided by
our paper. We feel our resource opens new lines of investigation into the biology of cell division in
the tissue context, where the effect of division on neighboring cells via adhesion and cell-cell
communication is an interesting part of the problem.
Reviewer #3:
The revised manuscript presents increased biological validation of the resource by following on
two identified proteins. Preliminary data on carboxyl-targeting capture reagent was removed.
Removal of the carboxyl-targeting capture reagent has improved the manuscript in clarity. These
data were clearly preliminary and added therefore little to the main point of the manuscript.
PCDH7 is now presented as a cell-surface marker of mitotic Hela and MCF7 cells. Furthermore,
this establishes an experimental workflow for the comparison of interphase and mitotic cells that
may ultimately lead to the analysis of primary cells. This points towards interesting avenues of
cancer research that are appearing more possible with this work coming out.
For clarity and transparency the proteomics data should be described in completion, a pie chart
of all data should be shown with the slice that the remainder of the manuscript focuses on (GObased membrane) being highlighted. Possibly even more effective, a Venn diagram could show in
one circle the proteins detected in this study and in another those in GO marked as membrane
associated; the overlap would then be the ones followed further here.
We thank the reviewer for this constructive comment. In response we have revised the figures and
the tables, incorporating all the suggestions of the reviewer, as detailed below. We feel these clarify
the manuscript and make it more accessible to the broad audience we hope will be interested.
A new figure, "Figure 1D" added to the manuscript which represents the venn diagram of all
identified proteins and the proteins used in the analysis as suggested by the reviewer. Their GObased distribution is also shown (please see Figure 1D).
The GO-based focusing does not appear in the current figure 1 and therefore -and also because
of non-consistent wording- remains misleading. Not even Supplementary Table 1 lists the actual
number of identified and quantified proteins.
In the revised figure 1, Go-based focusing is highlighted. Revised Supplementary Table 1 includes
actual number of identified proteins.
GO-based filtering is an essential step of the actually used (but not in the presented) experimental
procedure and must feature in the flow chart, Figure 1C. Figure 1C is misleading as it suggests
that all results of the proteomic analysis are discussed in the manuscript and shown in Figure 2A
while it is actually a filtered subset.
In the revised Figure 1C, workflow had an extra step which shows the GO-based filtering step and it
is stated that the filtered data is further analyzed in Figure 2A.
Supplementary Table 4 should have a column stating if a protein group was included or not in the
analysis. The table also misses information needed to reanalyze it and obtain the same results as
the authors. Given the focus on resource this is essential.
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Supplementary Table 4 is revised and an extra column is added "Column B". The proteins those
were included to the cell surface analysis are indicated as "YES" and those were excluded are
indicated as "NO" for all proteins identified in 5 biological replicates.
P4: "mitotic proteins" proteins enriched in mitosis. To name them mitotic proteins a function
would need to be demonstrated in mitosis. Same for "interphase proteins".
"mitotic proteins" were corrected as "mitosis selective proteins" and "interphase proteins" were
corrected as "interphase selective proteins"
P5: Reduced proteomic detection of PCDH7 in interphase does not necessarily suggest
localization of this protein in an internal compartment in interphase.
We agree that reduced proteomic detection of PCDH7 in interphase may not be due to localization
of this protein in an internal compartment in interphase. However we make this decision by
combining the western-blot analysis result, which clearly shows that total PCDH7 protein
expression was not different in mitosis versus interphase lysates (Figure 2B), therefore it was
suggested that most PCDH7 is sequestered in an intracellular compartment during interphase.
Proteomic analysis bases on the protein being accessible to labeling. The methods section is not
clear on whether the HeLa S3 cells were grown in suspension or on plates.
We apologize for the omission, in the current method section we indicate that Hela S3 cells are
grown on plates. There were two reasons that we used Hela S3 cells as adherent rather then
suspension: 1. It was more practical to wash off dead cells and debris in the adherent cells than
suspension cells. 2. In suspension interphase cell morphology is also similar to mitotic cells with
round cells by comparing adhere interphase cell and less adhere mitotic cells we aim to identify
more proteins involve in the cell cycle dependent adherence. In order to maximize the accessibility
of labeling on the plates cells were labeled in 60-80% confluency.
Was intracellular PCDH7 only quantified for overexpressed GFP-labeled protein? What happens
when using fixed cells and antibodies against the native protein?
The intracellular PCDH7 was not only quantified for overexpressed GFP-labeled protein, Figure 3B
shows the quantification of fixed cell and antibodies against the anti-PCDH7 antibody, shown in
Figure 3A which clearly shows the mitotic selective cell surface expression of PCDH7.
P13: Figure 3A left/right presumably refers to Figure 2. "all proteins"? Is this really true, all
proteins identified in the five replica or only the subset obtained by GO-based filtering? The
details given should allow a reader to reconstruct the findings based on Supplementary Table 4!
We thank the reviewer for the input; following corrections are made in the revised version:
"Heavy-to-light ratios were computed and GO-based filtered cell membrane protein groups were
used for the subsequent analysis and transformed to a binary logarithmic scale. Since significant
variation between experimental repeats was observed as shown in Figure 2A left, all repeats were
normalized. Quantile normalization (Bolstad et al., 2003), an established standard technique from
the analysis of microarrays, was applied to all repeats and reduced experimental variation. The
resulting histogram of the joint distribution is shown in Figure 2A right."
All the details are given in the revised Supplementary Table 4.
Author response letter: "The sentence is changed as: A kinesin-5 inhibitor was chosen for mitotic
arrest because it leaves microtubule dynamics intact; in this respect it generates a mitotic arrest
that is more similar to normal metaphase than anti-microtubule drugs." I cannot confirm this.
Based on the reviewer's comment the sentence changed as below and a reference was included
which shows that STLC does not affect microtubule dynamics (see Figure 5 of Skoufias et al.,
2006).
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2013-85162
"A kinesin-5 inhibitor was chosen for mitotic arrest because in contrast to anti-microtubule drugs it
leaves microtubule dynamics intact (Skoufias et al., 2006)".
Accept letter
05 November 2014
Thank you very much for your patience and our apologies for the substantial delay in the re-evaluation of your
resource manuscript on mitotic cell surface proteomes.
Dr. Pulverer, our Chief Editor, and I have now together carefully assessed your answers and revisions added in
response to the previous reviewer comments and to our previous decision letter. I am pleased to inform you that
we have decided to accept your manuscript for publication in The EMBO Journal. pending some final minor
revisions concerning data presentation.
This presentational issue relates to the quality of the Western Blot panels in the figures. Thanks to
the source data you kindly provided, we are happy with these data in principle, however some of the
figures still do not represent the original data in a sufficiently faithful and direct manner. In
particular, the blots in Figure 2 appear very much over-adjusted for brightness and contrast, partially
distorted in shape compared to the original data, and lacking clear separation marks between the
individual rows. Therefore, please remake Figures 2B and 2C from the provided source data, with
clear separations and only moderate adjustment in order to maintain a positive correlation of the
electronic figure to the original blot data. In addition, I feel that also the 'Actin' panel in Figure 4D
should be remade, as it looks clearly more pixelated/solarized and over-contrasted compared to the
provided source data. To simplify things, you may simply send us these revised figures via email.
Once we should have received these final modifications from you, we should then hopefully be able
to swiftly proceed with formal acceptance and production of this resource article!
© European Molecular Biology Organization
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