The EMBO Journal Peer Review Process File - EMBO-2010-75926
Manuscript EMBO-2010-75926
Ca2+ induces clustering of membrane proteins in the plasma
membrane via electrostatic interactions
Felipe E. Zilly, Nagaraj D. Halemani, David Walrafen, Luis Spitta, Arne Schreiber, Reinhard Jahn
and Thorsten Lang
Corresponding author: Thorsten Lang, LIMES Institute
Review timeline:
Submission date:
Editorial Decision:
Resubmission:
Editorial Decision:
Revision received:
Editorial Decision:
Revision received:
Accepted:
06 August 2009
05 October 2009
07 September 2010
18 October 2010
06 January 2011
28 January 2011
01 February 2011
02 February 2011
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
05 October 2009
Thank you for submitting your manuscript for consideration by The EMBO Journal. Let me first of
all apologise for the exceptionally long delay in getting back to you with a decision. Unfortunately,
we experienced difficulties in finding suitable and willing referees for this manuscript. In addition
one of the referees was not able to get back to us as quickly as initially expected, and in fact we have
still not received his/her report.
Given that we have received a positive report (referee 1) as well as a negative report (referee 2; see
below) I have now also consulted with an expert editorial advisor of suitable expertise who knows
both the field and the journal very well. As you will see, referee 1 would be positive regarding
publication of the paper here after appropriate revision. More specifically, he/she feels strongly that
the effect of magnesium ions versus calcium ions needs to be analysed in some depth. Referee 2 is
clearly not in favour of publication of the manuscript here. This becomes even more explicit in
his/her overall rating returned to the editorial office. I will not repeat all his/her specific concerns
here, but he/she essentially feels that the study is too premature at present and that a considerably
stronger case for the physiological significance of your findings would be required (concerns
regarding the approach of using membrane sheets; concerns regarding the calcium concentrations
used). Furthermore, he/she feels that in terms of the functional significance of your findings the
study should be extended beyond SNARE proteins. Our expert editorial advisor also brings up the
issue of physiological significance. He/she feels that all the experiments (at least controls in the in
vitro experiments) should have been/should be performed in the presence of physiological
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magnesium concentrations. Furthermore, he/she feels that data should be provided that analyse how
the membrane organisation recovers from a physiological calcium influx. All in all it becomes clear
that you would need to make a stronger case for the physiological (and functional) significance of
your findings before the referee 2 (and our expert editorial advisor) would offer sufficient support
for publication of your paper here. So, quite an amount of additional experimentation would be
needed to address these concerns in an adequate manner, and the outcome of such experiments
cannot be predicted at this point. In such a situation, I am sorry to say that we cannot consider (and
thus commit to) a revision and I therefore see little choice but to come to the conclusion that we
cannot offer to publish the manuscript at this point.
Still, given the interest expressed by referee 1 and our editorial advisor in principle we would not
exclude to consider a substantially more developed version of this manuscript as a new submission.
However, such a new version of the manuscript would need to be treated as a new submission rather
than as a revision and evaluated again at the editorial level and reviewed afresh, also in the light of
the state of the literature at the time of resubmission. I should add that given the situation of having
conflicting referee reports at this point and given that referee 2 is clearly not in favour of publication
of the study here we will not only involve the two original referees in assessing the new manuscript
(if available), but we will need to involve a new independent reviewer. We feel that this will ensure
a fair assessment of a new submission.
Thank you in any case for the opportunity to consider this manuscript. I am sorry we cannot be more
positive on this occasion, but we hope nevertheless that you will find our referees' comments
helpful.
Yours sincerely,
Editor
The EMBO Journal
-----------------------------------------------REFEREE COMMENTS
Referee #1 (Remarks to the Author):
The paper reports interesting new measurements: calcium ions induce local clustering of proteins on
the plasma membrane. In my opinion the report is important and definitely worthy of publication
after minor revision.
I have two (both really quite minor) objections to the wording of particular sentences in the report.
The first is concerned with the conclusion on page 13 that "calcium directly acts on membrane
proteins via unspecific and fast electrostatic mechanisms."
If the effects produced by calcium ions are "unspecific", then similar concentrations of magnesium
ions (and other divalent alkaline earth cations) should produce similar clustering effects? The
authors do not mention experiments with other divalent cations. (Or if they did I missed it.) In a cell,
magnesium ions are thought to be present at a free concentration of about 1 µM. This is in contrast
to calcium ions, which are present at a free concentration of about 100 nM. Some of the effects they
observe occur with a calcium concentration of 0.75 uM (page 6), and others at 50 uM (page 6). I
suspect 1 and 50 uM magnesium ions do not produce the effects they observe with calcium ions?
The authors might want to do a few control experiments with magnesium ions. If magnesium ions
do not exert a similar effect, they might want to modify claim on page 13 about the 'unspecific'
nature of the effects they observe with calcium ions?
I agree with the suspicion of the authors that the calcium ions are probably acting on proteins rather
than lipids. Calcium, magnesium and other divalent cations bind with similar very weak affinities to
lipids commonly found on the inner leaflet of the plasma membrane. For example, they exert similar
effect on the electrostatic potential next to a phosphatidylcholine/phosphatidylserine (PC/PS)
membrane, provided the membranes/vesicles do not aggregate (e.g. J. Gen. Physiol. (1981) 77 445).
The effects of alkali metal and alkanline earth cations on these lipid membranes can indeed by
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described by electrostatic theory (Annu Rev. Biophys. Biophys. Chem. 1989. 18:113-36.). That is,
no significant binding to PS membranes is observed with calcium ions at concentrations < 100 uM
(in physiological monovalent concentration).
However, when calcium ions are allowed to cause aggregation of the PS vesicles, the affinity is
markedly increased (from weaker than µM to stronger than uM), as is the specificity for calcium
over magnesium ions (from ~two-fold to >thousand-fold). The mechanism of this high affinity, high
specificity binding of calcium ions to aggregated lipid membranes is still not understood, in spite of
much solid work by investigators such as G. W. Feigenson at Cornell. The hydration/dehydration
properties of Ca vs Mg are presumably important for the ability of calcium ions to cause the
aggregation and restructuring of PS membranes (presumably not a phenomenon of direct biological
importance). A similar involvement of the hydration/dehydration properties of Ca vs Mg might be
important for the Ca-induced aggregation of proteins on the plasma membrane observed by the
authors? In any case, I suspect their phenomenon involves more than just unspecific electrostatics
even though the binding sites might be comprised of simple clusters of acidic residues on the
proteins, as the authors suggest. If the effects seen by the authors are specific for calcium vs
magnesium ions, the authors might want to delete 'unspecific' and add an additional sentence or
phrase to point out that some specific chemical or hydration vs unspecific electrostatic properties of
the calcium ion might be involved?
A second minor comment concerns the claim in the last line of page 4 that other authors have
suggested "Ca2+ binding to PIP2 followed by release of peripherally associated positively charged
proteins" . The experiments I am aware of show that Ca2+ ions bind only weakly to PIP2 under
physiological conditions in an isolated membrane (but may 'bridge' lipids to proteins).
Referee #2 (Remarks to the Author):
At present this paper is not suitable for publication in EMBO J.
There are three main weaknesses:
1. Much of the data is based on experiments using membrane sheets. Preparation of these sheets
may change many physical and chemical properties of the plasma membrane as well as allowing
alteration of calcium concentrations. The authors have not carried out the important control of
analyzing protein distribution in membrane sheets under conditions where free calcium is carefully
buffered at the basal cytoplasmic concentration appropriate for the cells being studied. This will
allow more confident discrimination between calcium-specific and other effects on membrane
protein distribution in membrane sheets.
2. Much of the data is based on comparison of 'no' calcium / high calcium. A more relevant
comparison would be between different physiological calcium levels.
3.It is not clear to what extent the phenomena reported are SNARE-specific. In the only experiments
looking at intact cells only SNARE proteins are examined. SNARE distribution may be affected by
Ca2+ transients for good functional reasons in these cells, but the authors' want to interpret the data
as supporting a general mechanism for induction of protein aggregation. What happens to transferrin
receptor, other membrane proteins when chromaffin cells are depolarised ?
Resubmission
07 September 2010
Reviewer #1
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We would like to thank this reviewer very much for his/her thoughtful comments, interesting
suggestions and the in general positive attitude regarding recommendation for publication. He/she
raised several issues for our consideration which have been well taken, leading to a significant
improvement of the manuscript:
Referee #1 (Remarks to the Author):
The paper reports interesting new measurements: calcium ions induce local clustering of proteins
on the plasma membrane. In my opinion the report is important and definitely worthy of publication
after minor revision.
I have two (both really quite minor) objections to the wording of particular sentences in the report.
The first is concerned with the conclusion on page 13 that "calcium directly acts on membrane
proteins via unspecific and fast electrostatic mechanisms."
If the effects produced by calcium ions are "unspecific", then similar concentrations of magnesium
ions (and other divalent alkaline earth cations) should produce similar clustering effects? The
authors do not mention experiments with other divalent cations. (Or if they did I missed it.) In a
cell, magnesium ions are thought to be present at a free concentration of about 1 µM. This is in
contrast to calcium ions, which are present at a free concentration of about 100 nM. Some of the
effects they observe occur with a calcium concentration of 0.75 µM (page 6), and others at 50 µM
(page 6). I suspect 1 and 50 µM magnesium ions do not produce the effects they observe with
calcium ions? The authors might want to do a few control experiments with magnesium ions.
The referee is correct in pointing out this important issue. We have added Fig. S1, showing that at a
concentration of 2 µM MgCl2 remodelling of membrane proteins does not occur. This is in line with
our assumption that electrostatics is the underlying mechanism, as the dehydration enthalpy of Mg2+
is much higher when compared to Ca2+, and therefore Mg2+ binding to a negatively charged amino
acid would consume more energy. This argument has been incorporated by adding a new paragraph
to the discussion (last paragraph of the discussion on page 13).
If magnesium ions do not exert a similar effect, they might want to modify claim on page 13 about
the 'unspecific' nature of the effects they observe with calcium ions?
We agree that the term unspecific might be misinterpreted. Though the mechanism is in a way
unspecific as it does not discriminate between proteins, one could argue that Ca2+ may be the only
biologically relevant ion which is sufficiently positively charged to undergo strong ionic interactions
and at the same time allows dehydration, and therefore acts specifically compared to other ions. To
avoid any misinterpretation, we have changed the above mentioned sentence "calcium directly acts
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on membrane proteins via unspecific and fast electrostatic mechanisms" to "calcium directly acts on
membrane proteins via fast electrostatic mechanisms" (page 12, last sentence of top paragraph).
I agree with the suspicion of the authors that the calcium ions are probably acting on proteins
rather than lipids. Calcium, magnesium and other divalent cations bind with similar very weak
affinities to lipids commonly found on the inner leaflet of the plasma membrane. For example, they
exert similar effect on the electrostatic potential next to a phosphatidylcholine/phosphatidylserine
(PC/PS) membrane, provided the membranes/vesicles do not aggregate (e.g. J. Gen. Physiol. (1981)
77 445). The effects of alkali metal and alkanline earth cations on these lipid membranes can
indeed by described by electrostatic theory (Annu Rev. Biophys. Biophys. Chem. 1989. 18:113-36.).
That is, no significant binding to PS membranes is observed with calcium ions at concentrations <
100 uM (in physiological monovalent concentration).
However, when calcium ions are allowed to cause aggregation of the PS vesicles, the affinity is
markedly increased (from weaker than µM to stronger than uM), as is the specificity for calcium
over magnesium ions (from ~two-fold to >thousand-fold). The mechanism of this high affinity, high
specificity binding of calcium ions to aggregated lipid membranes is still not understood, in spite of
much solid work by investigators such as G. W. Feigenson at Cornell. The hydration/dehydration
properties of Ca vs Mg are presumably important for the ability of calcium ions to cause the
aggregation and restructuring of PS membranes (presumably not a phenomenon of direct biological
importance). A similar involvement of the hydration/dehydration properties of Ca vs Mg might be
important for the Ca-induced aggregation of proteins on the plasma membrane observed by the
authors?
This is definitely the case and the argument has been incorporated (see our reply to one of the
previous points).
In any case, I suspect their phenomenon involves more than just unspecific electrostatics even
though the binding sites might be comprised of simple clusters of acidic residues on the proteins, as
the authors suggest. If the effects seen by the authors are specific for calcium vs magnesium ions,
the authors might want to delete 'unspecific' and add an additional sentence or phrase to point out
that some specific chemical or hydration vs unspecific electrostatic properties of the calcium ion
might be involved?
As mentioned above, the term “unspecific” has been deleted and the paragraph added to the
discussion explains why Ca2+ is probably the only divalent ion capable of mediating this type of
electrostatic mechanism.
Comment
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A second minor comment concerns the claim in the last line of page 4 that other authors have
suggested "Ca2+ binding to PIP2 followed by release of peripherally associated positively charged
proteins". The experiments I am aware of show that Ca2+ ions bind only weakly to PIP2 under
physiological conditions in an isolated membrane (but may 'bridge' lipids to proteins).
We would like to thank the referee for this comment. We have removed this incorrect argument and
added “for other types of electrostatic interactions between proteins and PIP2 controlled by Ca2+ see
McLaughlin and Murray, 2005” (page 5, top).
Reviewer #2
We would also like to thank this reviewer very much for his/her more critical comments. The raised
issues were well taken and addressed as follows:
Referee #2 (Remarks to the Author):
At present this paper is not suitable for publication in EMBO J.
There are three main weaknesses:
1. Much of the data is based on experiments using membrane sheets. Preparation of these sheets
may change many physical and chemical properties of the plasma membrane as well as allowing
alteration of calcium concentrations. The authors have not carried out the important control of
analyzing protein distribution in membrane sheets under conditions where free calcium is carefully
buffered at the basal cytoplasmic concentration appropriate for the cells being studied. This will
allow more confident discrimination between calcium-specific and other effects on membrane
protein distribution in membrane sheets.
This experiment has been now added (new Fig. 2D). In addition, we have tested the effect of Mg2+
ions (new Fig. S1, see also reply to referee #1).
2. Much of the data is based on comparison of 'no' calcium / high calcium. A more relevant
comparison would be between different physiological calcium levels.
See our reply to the previous point.
3. It is not clear to what extent the phenomena reported are SNARE-specific. In the only experiments
looking at intact cells only SNARE proteins are examined. SNARE distribution may be affected by
Ca2+ transients for good functional reasons in these cells, but the authors' want to interpret the data
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as supporting a general mechanism for induction of protein aggregation. What happens to
transferrin receptor, other membrane proteins when chromaffin cells are depolarised?
Compared to the old version of the manuscript, we have now tested all proteins and also reduced the
time of depolarisation to 30s, in order to avoid any artificially high intracellular Ca2+ rises and to
show that the effect is indeed very fast. As shown in new Fig. 3, all proteins stain less well after
depolarisation. SNAP25 is strongly affected, in line with being the most Ca2+ sensitive protein, and
transferrin receptor is least affected, also in line with being one of the least sensitive proteins. Only
SNAP23 staining is less responsive than expected, and such imperfect correlation between
experiments shown in Figs. 2 and 3 could be explained by a non-uniform elevation of Ca2+ beneath
the plasma membrane affecting differentially distributed proteins (regarding their distance to
calcium channels) to a correspondingly varying extend (see also last sentence of the penultimate
paragraph on page 8).
In addition, in order to focus the manuscript to the general influence of calcium ions on membrane
structure and to avoid the impression that the study addresses SNARE-specific aspects, we have
moved Figs. 5 and 6 to the supplementary section (now Figs. S3 and S2, respectively).
Apart from the above mentioned changes, we felt that previous Fig. 4 displayed too many panels. To
make the messages clearer, we have removed some panels and show the different masking
mechanisms in two different figures (now Figs. 4 and 5).
Finally, we would like to thank once more the referees very much for their constructive comments
which helped to improve the manuscript significantly.
2nd Editorial Decision
18 October 2010
Thank you for submitting a new version of your original manuscript EMBOJ-2009-72129 as a new
submission. Let me first of all apologise for the exceptionally long delay in getting back to you with
a decision. Unfortunately, we experienced severe difficulties with the availability of our original
referee 1 and our original expert editorial advisor at this time. We therefore had to involve two new
referees (referee 2 and 3) in addition to our original referee 2 (now referee 1). We have now finally
received all three reports that are shown below.
As you will see referee 1 is still not convinced that you have made a sufficiently strong case for the
physiological significance of your findings. However, the other two referees are considerably more
positive and would support publication here, after toning down your conclusions and a number of
additional experiments. Given that we are treating this manuscript as a new submission rather than
as a revision I have come to the conclusion that we should be able to consider a revision in which
you need to address the referees' criticisms in an adequate manner. I should add that you should at
least respond to the issues raised by referee 1.
I should remind you that it is EMBO Journal policy to allow a single round of revision only and that,
therefore, acceptance or rejection of the manuscript will depend on the completeness of your
responses included in the next, final version of the manuscript.
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When preparing your letter of response to the referees' comments, please bear in mind that this will
form part of the Peer Review Process File, and will therefore be available online to the community.
For more details on our Transparent Editorial Process initiative, please visit our website:
http://www.nature.com/emboj/about/process.html
Thank you for the opportunity to consider your work for publication. I look forward to your
revision.
Yours sincerely,
Editor
The EMBO Journal
-----------------------------------------------REFEREE COMMENTS
Referee #1 (Remarks to the Author):
Zilly et al.
This paper uses isolated fragments of plasma membrane to study the effect of Ca2+ on membrane
protein distribution.
The paper is very weak on several different levels.
There is no evidence that the proposed change in protein distribution (which I do not believe occurs
in intact cells anyway - see below) is biologically important for anything. In order to constitute more
than a phenomenological curiosity, alteration in the distribution of one or more proteins in response
to Ca2+ would need to have a demonstrable effect on the function of that protein.
All of the main data in the paper are based on studying fixed cells. It is commonplace to note that
fixation may alter the distribution of proteins within the membrane at sub-micron distance scales.
This artefactual, aldehyde-induced clustering may well be sensitive to the amount of Ca2+ present.
This possibility is not adequately controlled for.
The paper also makes extensive use of membrane sheets. When the cytoplasm is blasted off a cell
membrane, one can expect that dozens of factors may change. PIP2 levels will alter, cortical
proteins may aggregate onto the membrane, ATP-dependent processes including maintenance of the
lipid asymmetry of the plasma membrane will cease, proteases may have access to previously
inaccessible substrates. Ca2+ ions may well modulate many of these non-physiological effects. The
authors state on p.7 that the fact that the effects that they report occur in fresh sheets exludes
artifacts. For example they state that this excludes 'proteolysis (since proteases are expected to be
washed away)'. How do they know? Expectation is not enough. Control experiments are needed.
The images of Synx1-GFP in figure 4 look the same with and without Ca2+. If there is any effect
here, it needs careful quantification to make it credible.
The authors propose that physiological fluctuations in cytoplasmic Ca2+ cause large-scale clustering
/ lateral re-distribution of membrane proteins. It is easy to add drugs to cells that increase
cytoplasmic Ca2+ using physiological signaling pathways and channels. It is easy to take TIR
images of the plasma membrane of intact, live cells expressing different GFP-tagged membrane
proteins during such perturbations. If the authors model is correct then they will observe a very
obvious and quantifiable redistribution of membrane proteins into clusters. It is easy to repeat the
experiment following depletion of intra and extra-cellular Ca2+ pools. This experiment would
address my concerns, and it is eminently do-able.
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Referee #2 (Remarks to the Author):
The authors investigated the role of calcium ions in membrane protein organization. The study
shows that elevated Ca2+ levels induce the rearrangement of membrane proteins in plasma
membrane sheets, but also in intact cells. The effect of the calcium on membrane protein
organization was studied by analysis of the fluorescence intensity distribution of antibodies bound to
different membrane proteins. Already in presence of nanomolar concentrations of calcium,
alterations in the immunostaining patterns were observed, as the signals appeared more clustered
and with a decreased fluorescence intensity. These changes in fluorescence distribution in presence
of Ca2+ were interpreted either as clustering of proteins in the membrane or conformational changes
of proteins, leading to masking of the antibody binding site. In order to distinguish these two
possibilities, control experiments were done on GFP and YFP tagged proteins. Distribution of these
proteins on the membrane also appeared to be more inhomogeneous in presence of elevated
levels of Ca2+ , but for some proteins was shown to differ from the immunostaining pattern.
In addition, the accessibility of protein binding sites to specific ligands, and therefore to antibodies,
were shown to be altered after Ca2+ treatment. These results support the hypothesis that Ca2+ leads
to protein clustering and causes conformational changes in the structure of protein. Both effects had
most probably electrostatic nature.
In general, the study addresses an important question and provides new insights in the role of
divalent cations in the organization of the cellular membrane, and possibly the
formation of membrane microdomains. As the research was done on membrane sheets lacking
cytosol, the membrane trafficking machinery and actin cortex, remodeling and clustering of
membrane proteins into possible microdomains just by Ca treatment represents a new and
interesting result.
However, some issues need to be addressed by the authors:
* As a main concern, the major part of the work is based only on qualitative observations. The only
quantitative measurement (FRAP) is summarized within one sentence. This experiment shows some
potential and should be more rigorously explored. Particularly, the authors should try to address the
question concerning the mobile fractions of the protein. As they claim that two distinct fraction
exist, they should also provide stronger arguments for that (like providing the diffusion coefficients
of these fractions).
* page 9: the given second reason for the remodeling of the membrane does not necessarily apply. It
might happen that conformational changes are already enough to mask the
antibody binding site. The protein need not be brought closer to the surface
* The analysis of fluorescence signals is not very clear. Which areas were taken for comparison of
fluorescent intensities?
* On figures 4 and 5 , for more precise comparison of the behavior of GFP and YFP tagged proteins
with that of immunolabeled proteins, the analysis of fluorescence intensities as it was done in
figures 2 and 3 would be appropriate
* It would be good to plot the dependence of fluorescence
intensity changes on Ca2+ concentration in figure 2. The same
plot could be also done in Fig.5 for antibody labeled SNAP 25, in
order to compare it to the plot presented on figure S2 for the
binding of Syb-594 to SNAP 25.
Referee #3 (Remarks to the Author):
The paper by Zilly et al. reports results which, on one hand, make sense, on the other hand, had been
envisaged only superficially and never investigated in detail. Therefore the paper is of interest and
might be the starting point for many other studies. The paper, however, reports only distribution, and
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possibly clustering results. Therefore, it has nothing to do with the functions of the proteins
investigated which, at the most, can be envisaged only hypothetically. This should be explicitly
stated in the paper. In addition, I have three problems that need additional experimental evidence to
be answered.
1. The authors say that the concentration of free Ca2+ in the cytosol is around 100 nM. This is true,
however on the average. Extensive work, especially by the group of Pozzan and Rizzuto, quoted in
the paper, has demonstrated that in the proximity of the plasma membrane, where the events
described in this paper occur, the concentration (Ca2+)p is considerably higher even at rest. The
cell-free results with plasma membranes should therefore be reconsidered with a new starting point;
those with intact cells require the correct value to be measured experimentally.
2. the changes in the distribution of the proteins are reported to be fast, however based on an indirect
criterion, their occurrence upon K+depolarization of the cells. The (Ca2+)p rise induced by K+
depolarization, however, is not so fast and especially, not so short-lived, much longer than those
induced by more physiological depolarizations. The, authors should reconsider the problem, which
is of key importance, and investigate the changes of protein organization in plasma membranes
exposed for very short time to buffers of precise (Ca2+). It is possible that the two processes
described, aggregation and decrease of fluorescence, have an independent time-course. In cse this is
indeed the case, it would reinforce the paper considerably.
3. A piece of evidence that is missing completely is whether the process is reversible when the
(Ca2+)p is dissipated, and in case how fast this reversion is. This is a critical point. Without this
information even hypotheses about the function of the aggregations would be inappropriate.
Finally, the title and Abstract. The first speaks about remodelling of membrane proteins.
Remodelling to me means that the structure of the proteins is changed, which is reasonable but is not
demonstrated here. The abstract, on the contrary, speaks only of microdomains.
I suggest to change the title into "Redistribution of various membrane proteins, from diffuse to more
clustered patterns, induced by Ca2+ via an electrostic mechanism". The Abstract should be rewritten in agreement with the title.
1st Revision - authors' response
06 January 2011
We would like to thank very much the referees for their critical evaluation of the manuscript and
constructive comments.
Referee #1 (Remarks to the Author):
Zilly et al.
This paper uses isolated fragments of plasma membrane to study the effect of Ca2+ on membrane
protein distribution.
The paper is very weak on several different levels.
There is no evidence that the proposed change in protein distribution (which I do not believe occurs
in intact cells anyway - see below) is biologically important for anything.
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To a certain extent, we understand the scepticism of this reviewer. In fact, our initial observations
date back more than 8 years, and for quite some time we considered them as experimental artefacts
not worthy of further investigation. However, with the observations being absolutely consistent in
every experiment we became more interested and added more and more data, using complementary
and independent approaches, which now convince us that the calcium-induced clustering of
membrane proteins is indeed a relevant and exciting finding. Of course, there are loose ends, in
particular as to which extent the calcium-dependent clustering affects protein function. With the
increasing evidence that lateral organization in the plane of the membrane is indeed highly relevant
for signalling (signalling platforms etc.), our findings may well be “the starting point for many other
studies” as pointed out by referee #2. For the SNAREs, at least, we have provided evidence that the
status of the proteins is altered. The relevance and general interest of our findings was clearly
acknowledged by referees #2 and #3, and also by the very positive original referee #1 who evaluated
our first version (in which the current referee #1 has been referee #2). Hence, altogether three out of
four referees appreciate the novelty and importance of the study.
In any case, as requested by the other two referees we have been more careful in the interpretation of
our data during revision in order not to be too speculative when discussing the biological importance
of the Ca2+ mediated redistribution. Accordingly, we have revised the second last paragraph of the
discussion on pages 15 - 16.
In order to constitute more than a phenomenological curiosity, alteration in the distribution of one
or more proteins in response to Ca2+ would need to have a demonstrable effect on the function of
that protein.
To stress that protein responses to Ca2+ affect their function, we have moved previous
Supplementary Figure S3 to the main section (now Fig. 6; see also new paragraph on pages 12 - 13).
It shows that acceptor complex formation between syntaxin 1A and SNAP25 is strongly inhibited by
Ca2+ in a dose dependent manner, documenting that disturbance of the clustering states affect the
ability of the proteins to undergo functionally relevant protein-protein interactions.
All of the main data in the paper are based on studying fixed cells. It is commonplace to note that
fixation may alter the distribution of proteins within the membrane at sub-micron distance scales.
This artefactual, aldehyde-induced clustering may well be sensitive to the amount of Ca2+ present.
This possibility is not adequately controlled for.
The referee probably refers to clustering artefacts occurring after delipidation with Triton X-100,
which is commonly used to open the cell membrane of fixed cells, allowing antibody access to the
antigens. In our membrane sheet experiments we do not apply Triton-X100, providing a better
morphology and stability of the membrane.
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However, to rule out that, as speculated by the referee, traces of Ca2+ present during fixation may
cause the observed effects, in the new sets of membrane sheet experiments we added EGTA to all
solutions applied after the Ca2+ treatments. As shown in new Figs. 3 and S3, no change was
observed (compare Fig. 2B and new data in Figs. 3 and S3), ruling out Ca2+ sensitive aldehydeinduced clustering being responsible for the observed effect.
In addition, we have added two more data sets in which the state of membrane proteins is analysed
in live cells. As shown in new Fig. 4 (see also new paragraph on pages 9 - 11), FRAP analysis
reveals Ca2+ dependent immobilisation of a fraction of membrane proteins and TIRF-microscopy
shows Ca2+ dependent quenching of GFP-fluorescence (Fig. S6).
The paper also makes extensive use of membrane sheets. When the cytoplasm is blasted off a cell
membrane, one can expect that dozens of factors may change. PIP2 levels will alter, cortical
proteins may aggregate onto the membrane, ATP-dependent processes including maintenance of the
lipid asymmetry of the plasma membrane will cease, proteases may have access to previously
inaccessible substrates. Ca2+ ions may well modulate many of these non-physiological effects. The
authors state on p.7 that the fact that the effects that they report occur in fresh sheets exludes
artifacts. For example they state that this excludes 'proteolysis (since proteases are expected to be
washed away)'. How do they know? Expectation is not enough. Control experiments are needed.
Obviously, the question as to which extent physiological conditions can be maintained in membrane
sheets is of high importance. We would like to point out that such membrane sheets are in use since
decades, e.g. in the classical work of John Heuser in imaging clathrin coated pits, caveolae, etc., and
our lab has extensive experience in using these preparations for functional studies (exocytosis and
endocytosis). Nevertheless, the referee is correct in that for every new observation made in this
preparation, confirmation in intact cells is necessary. As stated already above, this is exactly what
we have done, and we have expanded these experiments during revision (Figs. 4 and S6). Also, we
have added a cocktail of protease inhibitors to rule out that protease activity is causally involved in
the Ca2+ dependent decrease of immunostaining (new Fig. S3 and page 7, lines 10 - 11).
The images of Synx1-GFP in figure 4 look the same with and without Ca2+. If there is any effect
here, it needs careful quantification to make it credible.
We agree that in the previous Fig. 4 (now Fig. S4) it is difficult to appreciate the difference. To this
end we have now applied a presentation of data in Fig. 3 (new experiment added upon request of
referee #3; see also corresponding text on page 9, lines 14 - 18) in which images were normalized to
the mean fluorescence of the directly fixed membrane sheet, and shown at the same scaling. Using
this presentation it is obvious that the fluorescence originates from more clustered patterns upon
Ca2+ treatment. As requested by the referee, we have quantified the degree of protein clustering by
calculating the normalized standard deviation from the mean pixel intensity (Fig. 3C and F). In a
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perfectly uniform image (without any instrument noise) the S.D. would be 0, whereas it becomes
larger the spottier the signal becomes. Analysis was performed on the data set for Fig. 3, as here
membrane sheets are not generated from cells expressing additional syntaxin-GFP (as in Fig. S4),
making any comparisons difficult. As shown in Fig. 3C and F, the S.D. is stable under control
conditions, whereas it strongly increases after Ca2+ treatment.
The authors propose that physiological fluctuations in cytoplasmic Ca2+ cause large-scale
clustering / lateral re-distribution of membrane proteins. It is easy to add drugs to cells that
increase cytoplasmic Ca2+ using physiological signaling pathways and channels. It is easy to take
TIR images of the plasma membrane of intact, live cells expressing different GFP-tagged membrane
proteins during such perturbations. If the authors model is correct then they will observe a very
obvious and quantifiable redistribution of membrane proteins into clusters. It is easy to repeat the
experiment following depletion of intra and extra-cellular Ca2+ pools. This experiment would
address my concerns, and it is eminently do-able.
These are all reasonable suggestions, and we have taken up some of them. As discussed above, we
used FRAP analysis and TIRF imaging to document Ca2+ -induced clustering in living cells. We
have used depolarization as a physiological stimulus to increase cytoplasmic Ca2+ through calcium
influx via calcium channels in Figs. 5 and S7 (previous Fig. 3), all confirming our conclusions. Of
course, one can always do more but we strongly believe that we have provided a comprehensive
data set that constitutes a significant advance and is ready for publication in a major journal. As
stated, there are always loose ends but we strongly believe that they are indeed exciting subjects for
future work.
Referee #2 (Remarks to the Author):
The authors investigated the role of calcium ions in membrane protein organization. The study
shows that elevated Ca2+ levels induce the rearrangement of membrane proteins in plasma
membrane sheets, but also in intact cells. The effect of the calcium on membrane protein
organization was studied by analysis of the fluorescence intensity distribution of antibodies bound to
different membrane proteins. Already in presence of nanomolar concentrations of calcium,
alterations in the immunostaining patterns were observed, as the signals appeared more clustered
and with a decreased fluorescence intensity. These changes in fluorescence distribution in presence
of Ca2+ were interpreted either as clustering of proteins in the membrane or conformational
changes of proteins, leading to masking of the antibody binding site. In order to distinguish these
two possibilities, control experiments were done on GFP and YFP tagged proteins. Distribution of
these proteins on the membrane also appeared to be more inhomogeneous in presence of elevated
levels of Ca2+, but for some proteins was shown to differ from the immunostaining pattern.
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In addition, the accessibility of protein binding sites to specific ligands, and therefore to antibodies,
were shown to be altered after Ca2+ treatment. These results support the hypothesis that Ca2+ leads
to protein clustering and causes conformational changes in the structure of protein. Both effects had
most probably electrostatic nature.
In general, the study addresses an important question and provides new insights in the role of
divalent cations in the organization of the cellular membrane, and possibly the formation of
membrane microdomains. As the research was done on membrane sheets lacking cytosol, the
membrane trafficking machinery and actin cortex, remodeling and clustering of membrane proteins
into possible microdomains just by Ca treatment represents a new and interesting result.
We would like to thank the referee very much for his/her positive evaluation.
However, some issues need to be addressed by the authors:
* As a main concern, the major part of the work is based only on qualitative observations. The
only quantitative measurement (FRAP) is summarized within one sentence. This experiment shows
some potential and should be more rigorously explored. Particularly, the authors should try to
address the question concerning the mobile fractions of the protein. As they claim that two distinct
fraction exist, they should also provide stronger arguments for that (like providing the diffusion
coefficients of these fractions).
We would like to thank the referee very much for this very useful suggestion. We have now
explored the FRAP approach also for syntaxin and analyzed the data more in depth. In line with the
earlier observations made for SNAP25, we find that Ca2+ also increases the size of the immobile
syntaxin fraction. The size increase of the immobile fraction has been quantified and is discussed in
terms of providing additional evidence for our hypothesis that Ca2+ leads to an increase in the
clustering state. Regarding the diffusion coefficients, we would rather prefer not to provide values
for the mobile and immobile fraction, as in these experiments only the apparent diffusion coefficient
is determined for the mobile fraction and the immobile fraction should not diffuse. The new data is
shown in Fig. 4 and discussed in a new paragraph on pages 9 - 11.
* page 9: the given second reason for the remodeling of the membrane does not necessarily apply. It
might happen that conformational changes are already enough to mask the antibody binding site.
The protein need not be brought closer to the surface
We have rephrased the argument accordingly (page 8, lines 11 - 12).
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* The analysis of fluorescence signals is not very clear. Which areas were taken for comparison of
fluorescent intensities?
We apologize for not having made this clear enough. We have extended the methods section
describing in detail how fluorescence intensities were analyzed (page 22, last paragraph).
* On figures 4 and 5, for more precise comparison of the behavior of GFP and YFP tagged proteins
with that of immunolabeled proteins, the analysis of fluorescence intensities as it was done in
figures 2 and 3 would be appropriate
We have now plotted immunostaining intensity against GFP and YFP fluorescene. As shown in
Figs. S4C and S5C, at low expression levels Ca2+ decreases immunostaining intensity to the same
degree when compared to Fig. 2B or Fig. S3. In the case of syntaxin, starting from an offset
reflecting the endogenous protein, immunostaining increases linearly with the syntaxin expression
level. In the case of SNAP25 further expression leads to saturation effects, probably because
SNAP25 is already available in non-overexpressing cells at a very high surface density of 7500
molecules per µM2 (Knowles et al., 2010). These issues are discussed now quantitatively in the
figure legends of Figs. S4 and S5. In addition, we analysed the correlation between the fluorescent
protein and the immunostaining channel to obtain a quantifiable measure for the similarity of the
two channels. Values are discussed and given in the text (page 8-9) and the corresponding figure
legends.
* It would be good to plot the dependence of fluorescence intensity changes on Ca2+ concentration
in figure 2. The same plot could be also done in Fig.5 for antibody labeled SNAP 25, in order to
compare it to the plot presented on figure S2 for the binding of Syb-594 to SNAP 25.
We prefer not to show data from Figs. 2B, C and D in one plot, as in Fig. 2B also magnesium was
present but not in Figs. 2C and D. Regarding earlier Fig. 5 (now Fig. S5), we believe there is a
misunderstanding, as here only no and 54 µM Ca2+ were tested.
Referee #3 (Remarks to the Author):
The paper by Zilly et al. reports results which, on one hand, make sense, on the other hand, had
been envisaged only superficially and never investigated in detail. Therefore the paper is of interest
and might be the starting point for many other studies. The paper, however, reports only
distribution, and possibly clustering results. Therefore, it has nothing to do with the functions of the
proteins investigated which, at the most, can be envisaged only hypothetically. This should be
explicitly stated in the paper.
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We would like to thank the referee for his/her in general positive evaluation and his/her suggestion
to discuss more explicitly that only hypothetically protein distribution and function are related to
each other. To this end we have now toned down our conclusion and modified the second last
paragraph of the discussion on pages 15 - 16 (see also reply to referee #1).
In addition, to point towards the possibility that Ca2+ mediated redistribution may affect the
functionality of membrane proteins, we have moved previous Fig. S3 to the main section (now Fig.
6; see also reply to the second point of referee #1).
In addition, I have three problems that need additional experimental evidence to be answered.
1. The authors say that the concentration of free Ca2+ in the cytosol is around 100 nM. This is true,
however on the average. Extensive work, especially by the group of Pozzan and Rizzuto, quoted in
the paper, has demonstrated that in the proximity of the plasma membrane, where the events
described in this paper occur, the concentration (Ca2+)p is considerably higher even at rest. The
cell-free results with plasma membranes should therefore be reconsidered with a new starting point;
We would like to thank the referee very much for pointing out this important issue. We discuss in a
new paragraph in the discussion how the difference between average cytosolic and putatively higher
subplasmalemmal Ca2+ would affect our conclusions (page 15, starting at line 11).
those with intact cells require the correct value to be measured experimentally.
We agree that added valuable information would be the subplasmalemmal Ca2+ concentration in
depolarized chromaffin cells. We are aware of the literature that reports highly variable values
applying Ca2+ sensitive dyes, in some cases even measured at sites of presumably single channels. In
any case, these measurements are very difficult to perform and to interpret, in particular as the
spatial resolution of a microscope would allow only measuring the average of a steep gradient (see
also the above studies by Nagai et al., 2004, Isshiki et al. 2002 and Marsault et al., 1997 which are
cited now for addressing the first issue raised by referee #3). After all, the chromaffin cell
experiments are not used for relating the magnitude of the Ca2+ effect to the free concentration of
calcium, but rather for showing that in principle also Ca2+ influx through channels can mediate the
effect. For studying the effect of a defined Ca2+ concentration, we believe that the membrane sheet
experiments are more suitable as they allow exposure of the subplasmalemmal area to a defined
calcium concentration.
2. the changes in the distribution of the proteins are reported to be fast, however based on an
indirect criterion, their occurrence upon K+ depolarization of the cells. The (Ca2+)p rise induced by
K+ depolarization, however, is not so fast and especially, not so short-lived, much longer than those
induced by more physiological depolarizations. The, authors should reconsider the problem, which
is of key importance, and investigate the changes of protein organization in plasma membranes
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exposed for very short time to buffers of precise (Ca2+). It is possible that the two processes
described, aggregation and decrease of fluorescence, have an independent time-course. In cse this
is indeed the case, it would reinforce the paper considerably.
We have added the requested experiment incubating membrane sheets from 30 s to up to 10 min.
The experiments show that aggregation and decrease in fluorescence intensity are correlated (Fig. 3;
page 9, lines 14 - 18).
3. A piece of evidence that is missing completely is whether the process is reversible when the
(Ca2+)p is dissipated, and in case how fast this reversion is. This is a critical point. Without this
information even hypotheses about the function of the aggregations would be inappropriate.
We have now added an experiment showing the time course of recovery (new Fig. S1).
Finally, the title and Abstract. The first speaks about remodelling of membrane proteins.
Remodelling to me means that the structure of the proteins is changed, which is reasonable but is
not demonstrated here. The abstract, on the contrary, speaks only of microdomains.
I suggest to change the title into "Redistribution of various membrane proteins, from diffuse to more
clustered patterns, induced by Ca2+ via an electrostic mechanism". The Abstract should be rewritten in agreement with the title.
We have removed the words “remodelling” and “microdomains” from the title and changed the
abstract as requested by the referee.
Finally, we would like to apologize for a typing error (850 nM has been given as 750 nM) that has
been corrected.
3rd Editorial Decision
28 January 2011
Thank you for sending us your revised manuscript. Our referees 2 and 3 have now seen it again. In
general, the referees are now positive about publication of your paper. Still, they feel that there are a
few remaining issues that need to be addressed (see below) before we can ultimately accept your
manuscript. I would therefore like to ask you to address or respond to these points in an amended
version of the manuscript.
Furthermore, there is one editorial issue that needs further attention. I would like to ask you not to
include the supplementary material into the merged manuscript file, but to generate a combined
supplementary material file that includes the supplementary text, the table S1 and the labelled
supplementary figures.
We are looking forward to receiving the final version of the manuscript.
Yours sincerely,
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Editor
The EMBO Journal
-----------------------------------------------REFEREE COMMENTS
Referee #2 (Remarks to the Author):
The paper has overall improved, but still raises some issues.
Major points:
1. Regarding the FRAP experiments, the authors provided additional data including syntaxin.
Moreover, they supplemented the data with the fitted curves. In figure 4, they described the images
as a 1st FRAP and 2nd FRAP, which is confusing. It would be clearer for the reader to have
quantitative information there about the Calcium content.
2. In part B/C, the authors showed data points from their FRAP experiments. But the descriptions of
these plots are unclear. Do the plots show averages over 3-6 cells from one or more independent
experiments?
3. In part E/F, the authors should mention the exact formula that was used for fitting the data.
4. As apparently different sets of data was shown in 4C and 4F, there is some important discrepancy.
From the fit in 4F one can see that the recovery rate in both cases is almost the same, whereas in 4C,
the sample treated with Ca2+ recovers faster. This probably can be clarified by showing data for all
the samples. I would suggest to show the average of data points from all experiments with SD (with
the color coding used so far). As an addition to this, they should plot the black solid line of the fit
function. This would carry all the important information in one graph and add clarity. In case the
authors would like to present some examples of FRAP curves, I would suggest to move them to the
supplement.
5. The authors claim that diffusion of the mobile fraction increases after the treatment with Ca2+,
but they do not show any calculations or method on which they based their hypothesis. These data
could be quantitatively extracted from the fitting functions as diffusion coefficients and presented in
figure 4 as a column chart.
6. By knowing diffusion coefficients, it would be possible to look more carefully at the SNAP25 and
syntaxin fluorescence recovery. From 4F and 4C, one can see that the shape of the curves/recovery
rate in case of SNAP25 is almost the same, whereas in the case of syntaxin the overall shape is
altered by calcium ions (both, max recovery and recovery rate are changed). This may imply
different mechanisms of clustering in different protein species (transmembrane domains vs
palmitoyl-anchored proteins). The problem could be discussed more deeply then, which would
increase the significance of the work.
Referee #3 (Remarks to the Author):
The authors have appropriately revised the paper which is now greatly improved. Although the
approach cannot permit the authors to deal with its basic limitations as I already emphasized in the
first review, in my opinion the paper is now close to be acceptable. I recommend only two minor
changes
1. the timing of the experiments, even the 30 sec of SNAP25 is quite slow compared to the timing of
physiology in nerve cells. This should be acknowledged and emphasized. If indeed the time needed
for the clustering is so long, the risk is that the process is not physiological but occurs only when
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cells are suffering.
2. Ca2+ is reported both as such and as calcium. There is no apparent reason for this. In my opinion
the definition of calcium is appropriate only when the bound pool is taken into consideration. This
does not occur here. Therefore I strongly recommend to use Ca2+ everywhere.
2nd Revision - authors' response
01 February 2011
Referee #2 (Remarks to the Author):
The paper has overall improved, but still raises some issues.
Major points:
1. Regarding the FRAP experiments, the authors provided additional data including syntaxin.
Moreover, they supplemented the data with the fitted curves. In figure 4, they described the images
as a 1st FRAP and 2nd FRAP, which is confusing. It would be clearer for the reader to have
quantitative information there about the Calcium content.
We have removed the terms 1st FRAP and 2nd FRAP and replaced it by Ringer and Ringer/Ca2+carrier.
2. In part B/C, the authors showed data points from their FRAP experiments. But the descriptions of
these plots are unclear. Do the plots show averages over 3-6 cells from one or more independent
experiments?
The plots show averaged data from three independent experiments, and each of the independent
experiments is the average from 3 - 6 cells. In order to avoid any confusion we have revised the
figure legend and also show for overview the averaged fitted graphs in one plot with the averaged
data (see also reply to point #4).
3. In part E/F, the authors should mention the exact formula that was used for fitting the data.
We mention now the formula also in the figure legend.
4. As apparently different sets of data was shown in 4C and 4F, there is some important
discrepancy. From the fit in 4F one can see that the recovery rate in both cases is almost the same,
whereas in 4C, the sample treated with Ca2+ recovers faster. This probably can be clarified by
showing data for all the samples. I would suggest to show the average of data points from all
experiments with SD (with the color coding used so far). As an addition to this, they should plot the
black solid line of the fit function. This would carry all the important information in one graph and
add clarity. In case the authors would like to present some examples of FRAP curves, I would
suggest to move them to the supplement.
Actually we had plotted data for all samples (in Fig. 4C and 4F all data for syntaxin and SNAP25,
respectively), but the presentation of the data was not clear enough (see also reply to point #2). As
requested, we have now added also the fitted curves to Fig. 4C and F (now Fig. 4C and D), in order
to show all important information in one graph. The old Figures 4C and 4F have been moved to the
supplementary material (Fig. S6) as they illustrate the rescaling process that uses the post-bleach and
pre-bleach values as 0% and 100%, respectively.
5. The authors claim that diffusion of the mobile fraction increases after the treatment with Ca2+,
but they do not show any calculations or method on which they based their hypothesis. These data
could be quantitatively extracted from the fitting functions as diffusion coefficients and presented in
figure 4 as a column chart.
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We have added a column chart to Fig. 4 as requested (Fig. 4E). The determination of the diffusion
coefficients shows that Ca2+ increases the diffusion of syntaxin and SNAP25 by a factor of 4.2 and
1.8, respectively.
6. By knowing diffusion coefficients, it would be possible to look more carefully at the SNAP25 and
syntaxin fluorescence recovery. From 4F and 4C, one can see that the shape of the curves/recovery
rate in case of SNAP25 is almost the same, whereas in the case of syntaxin the overall shape is
altered by calcium ions (both, max recovery and recovery rate are changed). This may imply
different mechanisms of clustering in different protein species (transmembrane domains vs
palmitoyl-anchored proteins). The problem could be discussed more deeply then, which would
increase the significance of the work.
The referee is correct in his/her notion that Ca2+ has a stronger (2.3-fold) effect on the diffusion rate
of syntaxin when compared to SNAP25. We discuss this issue on page 11, line 18.
Referee #3 (Remarks to the Author):
The authors have appropriately revised the paper which is now greatly improved. Although the
approach cannot permit the authors to deal with its basic limitations as I already emphasized in the
first review, in my opinion the paper is now close to be acceptable. I recommend only two minor
changes
1. the timing of the experiments, even the 30 sec of SNAP25 is quite slow compared to the timing of
physiology in nerve cells. This should be acknowledged and emphasized. If indeed the time needed
for the clustering is so long, the risk is that the process is not physiological but occurs only when
cells are suffering.
The above argument has been stated in the discussion (page 16, line 14).
2. Ca2+ is reported both as such and as calcium. There is no apparent reason for this. In my
opinion the definition of calcium is appropriate only when the bound pool is taken into
consideration. This does not occur here. Therefore I strongly recommend to use Ca2+ everywhere.
As requested, we have replaced “calcium” by “Ca2+“ in the main manuscript and supplementary
material.
Finally, we would like to thank the referees for their helpful comments and excellent suggestions
during revision of the manuscript.
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