STIM1 and ORAI1 form a novel cold transduction mechanism in sensory and sympathetic neurons

Abstract Moderate coolness is sensed by TRPM8 ion channels in peripheral sensory nerves, but the mechanism by which noxious cold is detected remains elusive. Here, we show that somatosensory and sympathetic neurons express two distinct mechanisms to detect noxious cold. In the first, inhibition by cold of a background outward current causes membrane depolarization that activates an inward current through voltage‐dependent calcium (CaV) channels. A second cold‐activated mechanism is independent of membrane voltage, is inhibited by blockers of ORAI ion channels and by downregulation of STIM1, and is recapitulated in HEK293 cells by co‐expression of ORAI1 and STIM1. Using total internal reflection fluorescence microscopy we found that cold causes STIM1 to aggregate with and activate ORAI1 ion channels, in a mechanism similar to that underlying store‐operated calcium entry (SOCE), but directly activated by cold and not by emptying of calcium stores. This novel mechanism may explain the phenomenon of cold‐induced vasodilation (CIVD), in which extreme cold increases blood flow in order to preserve the integrity of peripheral tissues.

In this manuscript, Buijs and coworkers study the phenomenon of cold-induced calcium (Ca2+) increases in neurons, HEK293 and PC12 cells. Using pharmacological, fura-2 and electrophys approaches, the authors suggest that somatosensory and sympathetic neurons have evolved two distinct mechanisms of cold-induced cytosolic Ca2+ increases, both dependent on extracellular Ca2+: 1) via CaV channels and 2) via SOCE. The authors provide evidence that the SOCE-related mechanisms is dependent on STIM1 and Orai1, but not dependent on ER Ca2+ levels. Finally, the authors suggest that the phenomenon the describe may explain cold-induced vasodilation, but provide no experimental evidence for this.
I have several concerns that preclude publication of this manuscript in the current form: 1) Fluorescence is a temperature-dependent phenomenon. The authors should show how temperature separately affects the 340nm and 380nm Fura-2 signals (i.e. show some raw traces) to demonstrate that the ratios reported are not simply due to temperature-dependent fluorescence changes.
2) It is not clear why cold ramps were performed from 31 deg C to 4 deg C; what happens if the cold ramp starts at a more physiological temperature (i.e. 37 deg)?
3) With cells bathed in HEPES buffer, how can the authors exclude that the apparent temperature-dependent affects are not due to change in bathing medium pH given the deltapKa/deltaT of HEPES? 4) Page 10 -I don't think the appropriate figure is being referred to (should this be Fig 4d-g)? Similarly, further down that paragraph -should this be Figure 4d? 5) Figure 5b -the puncta formation due to cold is not convincing. It appears that those puncta are pre-existing at baseline. Further, the # of puncta per cell (panel b) does not seem to be consistent with the representative images shown in panel A. As a control, the authors should show how puncta form in response to ER Ca2+ depletion. 6) How can the authors reconcile that decreased number of STIM puncta at colder temperature with increased SOCE? Do the authors believe that the activation of Orai1 channels due to cold occurs independent of STIM1? If so, what would be the mechanism? 7) Previous work by Patapoutian group showed that increased temperatures (from 37 to above 40 deg C) induce STIM1 activation, and returning to lower temperatures causes profound activation of Orai1 channels, independent of ER Ca2+ levels. More specific discussion of this previous work and how it may related to the present observations is needed. 8) Previous work showed that nitric oxide suppresses SOCE and CRAC currents; how do the authors reconcile those previous observations with their supposition that the cold-induced SOCE drives CVID via nNOS activity? The authors should consider adding a model diagram with respect CVID signaling in the Discussion, which ties together all the observations from the present study and previous studies. Alternatively, a model diagram that ties together all previous temperature-induced cytosolic Ca2+ stimulation studies with the present observations would be very useful. 9) In general, the referencing in the manuscript appears to be dubious. There are several instances where the authors are referencing STIM1 and/Orai1 observations prior to 2005/2006, when in fact these proteins were not identified until 2005/2006. The authors need to carefully check all references and ensure they are referencing the primary publication. ** As a service to authors, EMBO Press provides authors with the possibility to transfer a manuscript that one journal cannot offer to publish to another EMBO publication or the open access journal Life Science Alliance launched in partnership between EMBO Press, Rockefeller University Press and Cold Spring Harbor Laboratory Press. The full manuscript and if applicable, reviewers' reports, are automatically sent to the receiving journal to allow for fast handling and a prompt decision on your manuscript. For more details of this service, and to transfer your manuscript please click on Link Not Available. ** Please do not share this URL as it will give anyone who clicks it access to your account.

Reply to: EMBOJ-2022-111348 Decision Letter "STIM1 and ORAI1 form a novel cold transduction mechanism in peripheral sensory and sympathetic neurons"
Referee #1: The manuscript entitled, "STIM1 and ORAI1 form a novel cold transduction mechanism in peripheral sensory and sympathetic neurons" provides evidence that store-operated Ca2+ entry may be a cold sensor. The strongest evidence by far are the observations that STIM1 RNAi eliminates cold sensitivity in SCG neurons and the fact that STIM and Orai colocalize in HEK293 cells overexpressing the proteins in response to cold. However, I also have some major concerns that need to be reconciled. Perhaps the most serious issue is that STIM1 and Orai1 are expressed in every cell except for erythrocytes. As such, it is unclear why novel cold sensitivity observed in a neuronal subset could be accounted for by STIM1 and Orai1 expression. I recognized that this issue was brought up in the discussion, but it should be raised in the introduction and directly addressed within the study. Similarly, the fact that STIM1 activation was previously shown to occur in response to heating and cooling cells should be discussed in the introduction and not at the end, as it is directly relevant to whether or not STIM/Orai are temperature sensitive. The extent to which the current findings reflect a new function of STIM1 should also be addressed.

Textual changes to address these comments will be made
Major Comments: 1. Although YM58483 does block Orai1, it blocks TRPC channels at similar concentrations (He et al, 2005) and activates TRPM4 at much lower concentrations (Takezawa et al, 2006). As such, these data are insufficient to demonstrate that Orai1 is cold-activated. Although I recognize and acknowledge that MRS1845 was also used. However, I have not seen any recent papers using this pharmacological agent; irrespective I am not aware of ANY truly specific inhibitors of Orai1.

We agree that pharmacological evidence is always open to the criticism that effects could be offtarget and for this reason we used two blockers of different structure that have both been shown in previous studies to inhibit Orai1. However, as made clear in the MS, we take the pharmacological evidence only as a guide to future studies in which downregulation of STIM1 in neurons and measures using TIRF microscopy of translocation of STIM1 and formation of ORAI1
puncta provide much stronger evidence for the involvement of STIM1 and ORAI1. We will make textual changes to reinforce this point.
2. The STIM1 siRNA experiments in fig 4 are crucial components of this study. Although somewhat convincing, the use of a scrambled control is necessary to show that this is not a transfection artifact.
We will perform further experiments in which the requested scrambled controls will be carried out.
3. The authors seem a little confused about where STIM1 is located in cells based on writing in both the introduction and in the description of figure 5. Although there is a small percentage of STIM1 in the plasma membrane, store depletion does not cause it to translocate from the ER to the PM.

24th May 2022 1st Authors' Response to Reviewers
Rather, it moves within the ER to areas of close ER-PM apposition. There are numerous primary articles and reviews establishing this point.

We are not confused but we accept that the text could be clearer. Our understanding coincides with that of the referee and we will make significant textual changes to clarify.
To that point, it is notable that STIM1 relocalization in response to cold is to areas where Orai1 is also moving to. In truth, these sites are ultimately controlled by STIM1, not Orai1, since STIM1 localization is space limited to these ER-PM junctions while Orai1 is free to diffuse through the PM. TIRFM can be done at many levels; these images suggest to me that this is a relatively wide TIRF image. They are also highly suggestive that STIM1 is cold sensitive.
As in the previous point, we accept that the text could be clearer. Our understanding coincides with that of the referee and we will make significant textual changes to clarify.
4. Given the central claim of this paper, a clear and direct demonstration that cold does not cause emptying of ER Ca2+ stores is needed by comparing ER Ca2+ release using thapsigargin or caffeine.

We will carry out the experiments suggested by the referee and will present the results in a revised MS.
Minor Comment: 1. Bottom paragraph of page 10, fig 3 is referenced when fig 4 is intended.

We will correct.
Referee #2: In this manuscript, Buijs and coworkers study the phenomenon of cold-induced calcium (Ca2+) increases in neurons, HEK293 and PC12 cells. Using pharmacological, fura-2 and electrophys approaches, the authors suggest that somatosensory and sympathetic neurons have evolved two distinct mechanisms of cold-induced cytosolic Ca2+ increases, both dependent on extracellular Ca2+: 1) via CaV channels and 2) via SOCE. The authors provide evidence that the SOCE-related mechanisms is dependent on STIM1 and Orai1, but not dependent on ER Ca2+ levels. Finally, the authors suggest that the phenomenon the describe may explain cold-induced vasodilation, but provide no experimental evidence for this.
I have several concerns that preclude publication of this manuscript in the current form: 1) Fluorescence is a temperature-dependent phenomenon. The authors should show how temperature separately affects the 340nm and 380nm Fura-2 signals (i.e. show some raw traces) to demonstrate that the ratios reported are not simply due to temperature-dependent fluorescence changes.

Agreed -we will supply the requested traces. Note also that in answer to Referee 1 we will provide stronger evidence that cold does not cause release of calcium from internal stores.
2) It is not clear why cold ramps were performed from 31 deg C to 4 deg C; what happens if the cold ramp starts at a more physiological temperature (i.e. 37 deg)?
The starting temperature of 31° was chosen as it closely reflects the skin temperature. We will carry out the requested experiments at a higher temperature and will supply the data in a revised MS.
3) With cells bathed in HEPES buffer, how can the authors exclude that the apparent temperaturedependent affects are not due to change in bathing medium pH given the deltapKa/deltaT of HEPES?
We will supply the requested measurements of pH as a function of temperature. We will also repeat critical experiments using buffer that has lower ΔKa/ΔT 4) Page 10 -I don't think the appropriate figure is being referred to (should this be  Figure 5b -the puncta formation due to cold is not convincing. It appears that those puncta are pre-existing at baseline. (Fig. 5a, middle image and Fig. 5b, c). STIM1 fluorescence is also visible, because the TIRF image was intentionally deep and includes STIM1 in the ER close to the surface membrane. We will supply further explanation in the text to reassure the referee.

Diffuse ORAI1 fluorescence is visible before application of cold in Fig. 5a (left image), as expected, but as shown quantitatively in Fig 5b is mostly below threshold. The effect of cold is to aggregate the ORAI1 fluorescence into distinct bright puncta
Further, the # of puncta per cell (panel b) does not seem to be consistent with the representative images shown in panel A.

Fig 5b-d are averages taken from 44 cells in 8 different wells, and cells were selected randomly, not based on whether there are no puncta present at baseline.
As a control, the authors should show how puncta form in response to ER Ca2+ depletion.

We will carry out experiments to address puncta formation after store depletion by examining the effects of ER depletion by thapsigargin on STIM1 translocation and ORAI1 puncta formation.
6) How can the authors reconcile that decreased number of STIM puncta at colder temperature with increased SOCE?
In answer to this point, the most obvious effect of cold, as shown in Fig. 5, is:

1) to cause translocation of STIM1 towards the cell membrane; 2) to increase the number of ORAI1 puncta by causing aggregation of existing ORAI1 channels in the surface membrane; 3) To increase co-localization of STIM1 and ORAI1
As the referee points out, there is also a much smaller (10-fold less) decrease in the number of STIM1 puncta (Fig. S8). This small change is probably due to movement of some STIM1 out of the plane of focus. However this is clearly a minor effect compared to the large increase in STIM1-Orai1 co-localization (Fig. 5). We will make textual changes and will combine Fig S8 into Fig. 5

to make the results clear for both STIM1 and ORAI1.
Do the authors believe that the activation of Orai1 channels due to cold occurs independent of STIM1? If so, what would be the mechanism?
Our data is consistent with STIM1 translocation to regions of the ER adjacent to the surface membrane, where STIM1 causes ORAI1 puncta formation and consequent activation of ORAI1 channels, and we did not intend to imply the scenario suggested by the referee. We will spell this out more clearly. 7) Previous work by Patapoutian group showed that increased temperatures (from 37 to above 40 deg C) induce STIM1 activation, and returning to lower temperatures causes profound activation of Orai1 channels, independent of ER Ca2+ levels. More specific discussion of this previous work and how it may related to the present observations is needed.

Our suggestion is that a calcium increase caused by the opening of ORAI1 channels may activate NOS and thus release the vasodilator NO from neuronal terminals, inducing vasodilation. Physiological levels of NO that are required to cause vasodilation in vivo are low. The work referred to (we presume the paper by Gui et al, J Mol Biol 2018) showing that NO inhibits SOCE used a high concentration in vitro of the NO donor GSNO (250μM). While the referee raises an interesting point, we do not feel that this work demonstrates conclusively that our proposal is invalid.
The authors should consider adding a model diagram with respect CVID signaling in the Discussion, which ties together all the observations from the present study and previous studies. Alternatively, a model diagram that ties together all previous temperature-induced cytosolic Ca2+ stimulation studies with the present observations would be very useful.

We agree that a diagram would increase the accessibility of our work and will provide a suitable one.
9) In general, the referencing in the manuscript appears to be dubious. There are several instances where the authors are referencing STIM1 and/Orai1 observations prior to 2005/2006, when in fact these proteins were not identified until 2005/2006. The authors need to carefully check all references and ensure they are referencing the primary publication.

We have looked through all references and can only find one instance of a reference to I CRAC dating
to before the discovery of ORAI channels. We will check and amend all references carefully.

27th May 2022 2nd Editorial Decision
Dear Prof. McNaughton, Firstly, I would like to thank you for trusting the appeals process at EMBO Journal. I have read your point-by-point response to the authors carefully, and have also taken a fresh look at the referees' reports and the manuscript files.
The initial editorial decision was based on my opinion that you had failed fully to convince the referees that ORAI1 and STIM1 combine to signal noxious cold in neurons. This was possibly due to both a lack of mechanistic detail (as implied by referee #2point 6), and a bold hypothesis which set a high bar for your experiments to clear.
Having said this, I fully appreciate that a number of the referees' concerns can be addressed. I am also very impressed by the elegance of the sensory circuit you propose. I would like, therefore, to reverse my decision and invite you to address the referees' comments in a revised version of the manuscript. At this early stage, it is important that I stress that i) your revised manuscript must receive strong support from both referees if it is to be published in EMBO Journal, and ii) it is difficult for me to predict (based on your revision plan) whether this support will be forthcoming. I therefore urge you to add as much mechanistic detail to the manuscript as you can. Should you wish to discuss how best to proceed, I would be happy to meet with you on Zoom one afternoon next week (or the week after). Let me know and I will send a link.
I should add that it is EMBO Journal policy to allow only a single round of revision, and acceptance of your manuscript will therefore depend on the completeness of your responses in this revised version.
When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process, please visit our website: https://www.embopress.org/page/journal/14602075/authorguide#transparentprocess We generally allow three months as standard revision time, although this is more of a guideline than a deadline. As a matter of policy, competing manuscripts published during this period will not negatively impact on our assessment of the conceptual advance presented by your study. However, we request that you contact the editor as soon as possible upon publication of any related work, to discuss how to proceed. Should you foresee a problem in meeting this three-month deadline, please let us know in advance and we may be able to grant an extension.
Thank you for the opportunity to consider your work for publication. I look forward to your revision.

William
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Reply to: EMBOJ-2022-111348 Decision Letter
"STIM1 and ORAI1 form a novel cold transduction mechanism in peripheral sensory and sympathetic neurons" Referee #1: The manuscript entitled, "STIM1 and ORAI1 form a novel cold transduction mechanism in peripheral sensory and sympathetic neurons" provides evidence that store-operated Ca2+ entry may be a cold sensor. The strongest evidence by far are the observations that STIM1 RNAi eliminates cold sensitivity in SCG neurons and the fact that STIM and Orai colocalize in HEK293 cells overexpressing the proteins in response to cold. However, I also have some major concerns that need to be reconciled. Perhaps the most serious issue is that STIM1 and Orai1 are expressed in every cell except for erythrocytes. As such, it is unclear why novel cold sensitivity observed in a neuronal subset could be accounted for by STIM1 and Orai1 expression. I recognized that this issue was brought up in the discussion, but it should be raised in the introduction and directly addressed within the study.

For the reasons outlined in the existing Discussion, it is unlikely that there is any difference, at the genetic level, between the cold-sensitive STIM expressed in some DRG and SCG neurons and the cold-insensitive STIM1 expressed in others, which leaves post-translational modification (e.g. phosphorylation, RNA editing) as the most likely explanation. We now discuss these possibilities in the revised Discussion. We feel that this particular issue is best kept in the Discussion rather than the Introduction as it deals with a matter arising directly from the results in the paper.
Similarly, the fact that STIM1 activation was previously shown to occur in response to heating and cooling cells should be discussed in the introduction and not at the end, as it is directly relevant to whether or not STIM/Orai are temperature sensitive.

We have retained an abbreviated discussion of the work of the Xiao and Stucky groups in the Discussion, but we now also discuss these papers in the Introduction together with a brief survey of the exciting discoveries about the function of STIM and ORAI that have been made in the last 15 years.
The extent to which the current findings reflect a new function of STIM1 should also be addressed.

We have now inserted a sentence in the Discussion dealing with this: "These observations show that STIM1, in addition to its function as a sensor of calcium levels in intracellular calcium stores, is also able to sense cold and trigger activation of ORAI1 channels without a change in intraluminal calcium levels."
Major Comments: 1. Although YM58483 does block Orai1, it blocks TRPC channels at similar concentrations (He et al, 2005) and activates TRPM4 at much lower concentrations (Takezawa et al, 2006). As such, these data are insufficient to demonstrate that Orai1 is cold-activated. Although I recognize and acknowledge that MRS1845 was also used. However, I have not seen any recent papers using this pharmacological agent; irrespective I am not aware of ANY truly specific inhibitors of Orai1. (Zhu et al., 2021). However, as is made clear in the MS, we take the pharmacological evidence only as a guide to studies in which: (1) expression of STIM1 and ORAI1 are shown to recapitulate the cold-activated calcium influx (Fig.4); (2) downregulation of STIM1 in neurons is shown to suppress cold-activated calcium influx (Fig. 4) (Fig. 5). These experiments provide much stronger evidence for the involvement of STIM1 and ORAI1. We have made the following textual changes on pg 11 to reinforce this point: (Hou et al., 2012) Fig. 4A-C)." 2. The STIM1 siRNA experiments in fig 4 are crucial components of this study. Although somewhat convincing, the use of a scrambled control is necessary to show that this is not a transfection artifact. Supplementary Fig. S11). There was no effect of the scrambled siRNA on cold-evoked calcium entry. Now added to text on pg 12: Fig. 11). " 3. The authors seem a little confused about where STIM1 is located in cells based on writing in both the introduction and in the description of figure 5. Although there is a small percentage of STIM1 in the plasma membrane, store depletion does not cause it to translocate from the ER to the PM. Rather, it moves within the ER to areas of close ER-PM apposition. There are numerous primary articles and reviews establishing this point.

We are not confused but we accept that our previous text could be clearer. Our understanding coincides with that of the referee and we have made significant textual changes to clarify. Changes to the Introduction have been outlined above, and the text under "STIM1 and ORAI1 form puncta in response to cold" on pg 13 has been modified: "In order to evoke a surface membrane calcium influx in response to emptying of intracellular stores (a store-operated calcium influx, SOCE), STIM1 proteins located in the endoplasmic reticulum aggregate and migrate within the ER membrane to areas of close ER-plasma membrane apposition, where they interact with and open ORAI1 ion channels (reviewed in Qiu & Lewis, 2019; Prakriya & Lewis, 2015). "
To that point, it is notable that STIM1 relocalization in response to cold is to areas where Orai1 is also moving to. In truth, these sites are ultimately controlled by STIM1, not Orai1, since STIM1 localization is space limited to these ER-PM junctions while Orai1 is free to diffuse through the PM. TIRFM can be done at many levels; these images suggest to me that this is a relatively wide TIRF image. They are also highly suggestive that STIM1 is cold sensitive.

The TIRF microscope was set up with the laser beam close to the critical angle so as to image a deep section (c. 100nm) of membrane and juxta-membrane features such as STIM1 within ER membrane. We now provide further details in Methods together with changes in the main text.
4. Given the central claim of this paper, a clear and direct demonstration that cold does not cause emptying of ER Ca2+ stores is needed by comparing ER Ca2+ release using thapsigargin or caffeine. Supplementary Fig  13.

During application of a cold stimulus in the absence of external calcium there is no detectable release of intracellular calcium, and on restoration of external calcium the intracellular calcium level returns to its previous level with little or no overshoot. When thapsigargin is applied in the absence of external calcium there is also no detectable discharge of store calcium, which is a little surprising but may be due to the known sparsity of the ER in neurons. However, on restoration of normal extracellular calcium levels there is a significantly greater overshoot due to SOCE, as expected when stores have been discharged by thapsigargin. These experiments confirm that there is no detectable store discharge caused by the application of cold.
Minor Comment: 1. Bottom paragraph of page 10, fig 3 is referenced when fig 4 is intended.

Referee #2:
In this manuscript, Buijs and coworkers study the phenomenon of cold-induced calcium (Ca2+) increases in neurons, HEK293 and PC12 cells. Using pharmacological, fura-2 and electrophys approaches, the authors suggest that somatosensory and sympathetic neurons have evolved two distinct mechanisms of cold-induced cytosolic Ca2+ increases, both dependent on extracellular Ca2+: 1) via CaV channels and 2) via SOCE. The authors provide evidence that the SOCE-related mechanisms is dependent on STIM1 and Orai1, but not dependent on ER Ca2+ levels. Finally, the authors suggest that the phenomenon the describe may explain cold-induced vasodilation, but provide no experimental evidence for this.
I have several concerns that preclude publication of this manuscript in the current form: 1) Fluorescence is a temperature-dependent phenomenon. The authors should show how temperature separately affects the 340nm and 380nm Fura-2 signals (i.e. show some raw traces) to demonstrate that the ratios reported are not simply due to temperature-dependent fluorescence changes. Supplementary Fig S3.

Although there are significant changes to each individual fluorescence trace, the ratio trace is stable when a cold ramp is imposed in the absence of external calcium, in contrast to significant changes when external calcium is present. We show that any minor temperature-dependent changes in the ration trace are much smaller than any of the calcium influx mechanisms elucidated in the paper.
2) It is not clear why cold ramps were performed from 31 deg C to 4 deg C; what happens if the cold ramp starts at a more physiological temperature (i.e. 37 deg)?
The starting temperature of 31-32° was chosen as it closely reflects the skin temperature and is therefore more "physiological" than 37°. We have now carried out experiments at a starting temperature of 37° and the results are shown in Supplementary Fig. S2. There is no significant difference from the results obtained at a starting temperature of 32°.
3) With cells bathed in HEPES buffer, how can the authors exclude that the apparent temperaturedependent affects are not due to change in bathing medium pH given the deltapKa/deltaT of HEPES? Fig. S4) 4) Page 10 -I don't think the appropriate figure is being referred to (should this be Fig 4d-g)? Similarly, further down that paragraph -should this be Figure 4d?

We have carried out measurements of pH as a function of temperature in buffer as used in the paper. A larger change in pH has no significant effect on the responses of SCG neurons to cold (Supplementary
Thank you for pointing this out. All Fig references have been carefully checked. Figure 5b -the puncta formation due to cold is not convincing. It appears that those puncta are pre-existing at baseline.

We agree that some puncta (particularly STIM1) are pre-existing and we apologise for not making clear how the experiment was done and therefore the likely reason for this. In this experiment we had difficulties with cell movement apparently caused by the application of a cold temperature. The experiment was therefore carried out in nominal zero calcium that was found to immobilise the cells. The low external Ca caused some aggregation of STIM1 and to a lesser extent ORAI1 into puncta. We have added a further description to the appropriate section in Results and also in Methods.
For ORAI1, some weakly fluorescent puncta are observed at 35°C (see Fig. 5A, B, C). The effect of cold is to cause a significant aggregation of diffuse ORAI1 fluorescence into distinct bright puncta and an approximately 3-fold increase in the number of puncta (see Fig. 5A, middle image and new Fig. 5D,E which includes both STIM1 and ORAI1 data appropriately colour-coded, see below). Thus while there are some ORAI1 puncta visible at baseline, it is not correct to say that that the ORAI puncta are all pre-existing. (Fig. 5D,E) but rather an increased colocalization of STIM1 with ORAI1 (Fig. 5F).

The section in Results dealing with this experiment has been rewritten to make the above clear
Further, the # of puncta per cell (panel b) does not seem to be consistent with the representative images shown in panel A. Fig  5A may

not appear representative. We now show the data of Fig. 5D and E as individual cells in B and C to show the degree of variability among cells, which we hope clarifies the issue. The changes shown in D and E for ORAI1 are highly significant.
As a control, the authors should show how puncta form in response to ER Ca2+ depletion.

This is a reasonable request but we have been unfortunately been unable to perform this experiment in a reasonable time frame because of unavailability of the apparatus. The experiment in Fig. S13 provides a partial answer in that it shows that following ER depletion by thapsigargin SOCE is activated, a process that has in many different cell types been shown to be associated with the formation of ORAI1 puncta.
6) How can the authors reconcile that decreased number of STIM puncta at colder temperature with increased SOCE?
The small apparent decrease in STIM1 puncta in Fig. 5 Fig. 5A. This increases the number of ORAI1 puncta by causing aggregation of existing ORAI1 channels in the surface membrane (Fig. 5A -E), and increases co-localization of STIM1 and ORAI1 (Fig. 5F). Fig S8 into Fig. 5

to make the results clear for both STIM1 and ORAI1.
Do the authors believe that the activation of Orai1 channels due to cold occurs independent of STIM1? If so, what would be the mechanism?
No, our data is consistent with translocation of pre-existing STIM1 puncta (whose formation has been induced by the low calcium in which the imaging was done, see above) to regions of the ER adjacent to the surface membrane, where STIM1 causes ORAI1 puncta formation and consequent activation of ORAI1 channels. We did not intend to imply the scenario suggested by the referee and we have now rewritten the text to make this clear 7) Previous work by Patapoutian group showed that increased temperatures (from 37 to above 40 deg C) induce STIM1 activation, and returning to lower temperatures causes profound activation of Orai1 channels, independent of ER Ca2+ levels. More specific discussion of this previous work and how it may related to the present observations is needed.

See reply to Ref 1 above. We have inserted a discussion of the Patapoutian group work (Xiao et al, 2011) and a later paper from one of the original authors (Liu et al, 2019) into the Introduction. However, we note that these authors used elevated temperatures above the normal range and did not look at the effect of cold temperatures. Their work was also carried out in non-neuronal cells. Our work breaks new ground by showing a STIM1-ORAI1 mechanism is important in the responses of neurons to extreme cold temperatures.
8) Previous work showed that nitric oxide suppresses SOCE and CRAC currents; how do the authors reconcile those previous observations with their supposition that the cold-induced SOCE drives CVID via nNOS activity?

Our paper shows that the opening of ORAI1 channels in SCG and DRG neurons causes an intracellular calcium increase. In the Discussion we suggest that this increase may activate NOS and thus release the vasodilator NO from neuronal terminals, providing a molecular explanation for the phenomenon of cold-induced vasodilation (CIVD). Physiological levels of NO that are required to cause vasodilation in vivo are low. The work referred to (we presume the paper by Gui et al, J Mol Biol 2018), showing that NO inhibits SOCE, used a high concentration in vitro of the NO donor GSNO (250μM). While the referee raises an interesting point, we do not feel that this work demonstrates conclusively that our proposal is invalid.
The authors should consider adding a model diagram with respect CVID signaling in the Discussion, which ties together all the observations from the present study and previous studies. Alternatively, a model diagram that ties together all previous temperature-induced cytosolic Ca2+ stimulation studies with the present observations would be very useful.
We agree that a diagram would increase the accessibility of our work and we now provide a suitable one (Fig. 7) that is referred to in the Discussion. We have now received re-review reports on your submitted manuscript from both referees, which are attached below. As you will see, you have addressed most of their comments satisfactorily. However, some concerns remain. The majority of these can be addressed without further experimentation, but I would like you to consider whether the manuscript is weakened by the absence of controls for Figure 5 which were requested by Reviewer 2.
In addition, there are some remaining editorial points which need to be addressed. Would you therefore please: include a "Data availability" section, include a "Disclosure and competing interests statement" and remove your declaration from the Acknowledgements section, complete the contributor role in the CrediT section of our website. CrediT replaces our author contributions section and allows you to provide more detailed descriptions than previously. Include the data referred to on page 11 ("data not shown") in an Appendix Figure and re-label the Appendix Figures accordingly. Please also add scale bars to Figure 1A and Figure 1E.
Regarding the figure callouts, Figure 1A and 1B should be called out before Figure 1C, 1D before 1G; Figure 3H should be called out after Figure 3G, and callouts for Figure 1E, 1F and 6A are missing.
We encourage the publication of source data with the aim of making primary data more accessible and transparent to the reader. It would be great if you could provide me with a PDF or Excel file for each figure that contains the original, unprocessed key pieces of data used in the figures. The files should be labeled with the appropriate figure/panel number; further annotation could be useful but is not essential. These files will be published online with the article as supplementary "Source Data" files. We anticipate that their inclusion will make your work more discoverable and useable to scientists in the future. We include a synopsis of the paper (see http://emboj.embopress.org/). Please provide me with a general summary statement and 3-5 bullet points that capture the key findings of the paper.
We also need a summary figure for the synopsis. The size should be 550 wide by   Please check that the title and abstract of the manuscript are brief, yet explicit, even to non-specialists.
When assembling figures, please refer to our figure preparation guideline in order to ensure proper formatting and readability in print as well as on screen: https://bit.ly/EMBOPressFigurePreparationGuideline See also figure legend guidelines: https://www.embopress.org/page/journal/14602075/authorguide#figureformat IMPORTANT: When you send the revision we will require -a point-by-point response to the referees' comments, with a detailed description of the changes made (as a word file).
-a word file of the manuscript text.
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Further information is available in our Guide For Authors: https://www.embopress.org/page/journal/14602075/authorguide We realize that it is difficult to revise to a specific deadline. In the interest of protecting the conceptual advance provided by the work, we recommend a revision within 3 months (15th Dec 2022). Please discuss the revision progress ahead of this time with the editor if you require more time to complete the revisions. Use the link below to submit your revision: https://emboj.msubmit.net/cgi-bin/main.plex 1. The idea that STIM1 could translocate from pre-formed puncta to a different pre-formed puncta is difficult to reconcile with my prior understanding of what puncta are. "Puncta" in this context generally refers to ER-PM junctions which STIM1 translocates to. I'm not sure what the aggregates of STIM1 are in DRGs at normal temperatures (Fig 5), but I don't think that they are puncta.
2. The discussion includes some speculations about cell-specific post-translational modifications of STIM1 that could account for DRG-specific cold sensitivity. However, the observation that this could be replicated in HEK293 and PC12 cells is not consistent with that explanation. If one accepts these results as correct, this would have to be an intrinsic property of STIM1.
Referee #2: Overall, the authors have done a good job addressing most of my concerns. Nevertheless, there remains some inconsistencies in the puncta data that are confusing and should be addressed prior to publication, as I have detailed below.
Reviewer 2 -major comment/response 5 -The new # of puncta counted for individual cells (Fig. 5D) remains inconsistent with the representative images shown in 5A. I can count ~4-5 Orai1 puncta induced after cold exposure, but the numbers in panel 5D suggest ~32-42. Again, a control experiment is needed to show how puncta form (counts/sizes/colocalization with STIM) in response to TG or caffeine treatment in their system. Could the authors consider a collaboration to complete this experiment or perhaps an alternative to TIRF (i.e. confocal?).
Reviewer 2 -major comment/response 6 -If STIM1 are in pre-existing puncta due to the nominally Ca2+-free buffer used, why are they not coupled with Orai1? If the nominally-free Ca2+ does not cause the depletion of ER Ca2+ stores, what is causing the pre-existing STIM1 puncta, specifically when using the Ca2+-free buffer?
Reviewer 1 -major comment/response 4 -The inability to detect ER Ca2+ discharge after TG treatment would suggest to me that the stores are already depleted to some extent. The larger "overshoot" after TG treatment + external Ca2+ re-addition could be because TG binding to SERCA is irreversible and the magnitude/extent/persistence of SOCE activation is enhanced with TG. In my opinion, the response to this important comment (related to reviewer 2 comment/response 6) needs more careful thought.
Minor: Reviewer 1 -major comment/response 1 -These are not isoforms that are expressed -these are homologues/paralogues. This needs to be clarified.

Replies to points raised by Editor
The majority of (the points raised by the referees) can be addressed without further experimentation, but I would like you to consider whether the manuscript is weakened by the absence of controls for Figure 5 which were requested by Reviewer 2.

See reply to Referee 2 below.
In addition, there are some remaining editorial points which need to be addressed. Would you therefore please: include a "Data availability" section, -Done include a "Disclosure and competing interests statement" and remove your declaration from the Acknowledgements section, -Done complete the contributor role in the CrediT section of our website. CrediT replaces our author contributions section and allows you to provide more detailed descriptions than previously. -Not able to access website. Editorial staff have assured us that author contributions have already been entered into the EMBO J. website. Include the data referred to on page 11 ("data not shown") in an Appendix Figure and re-label the Appendix Figures accordingly.
-Done Please also add scale bars to Figure 1A and Figure 1E.

-Done
Regarding the figure callouts, Figure 1A and 1B should be called out before Figure 1C, 1D before 1G; Figure 3H should be called out after Figure 3G, and callouts for Figure 1E, 1F and 6A are missing.

-All corrected
We encourage the publication of source data with the aim of making primary data more accessible and transparent to the reader. It would be great if you could provide me with a PDF or Excel file for each figure that contains the original, unprocessed key pieces of data used in the figures. The files should be labeled with the appropriate figure/panel number; further annotation could be useful but is not essential. These files will be published online with the article as supplementary "Source Data" files. We anticipate that their inclusion will make your work more discoverable and useable to scientis ts i n the future.

-Done and .zip files attached
We include a synopsis of the paper (see http://emboj.embopress.org/). Please provide me with a general summary statement and 3-5 bullet points that capture the key findings of the paper. -Done We also need a summary figure for the synopsis. The size should be 550 wide by  high (pixels). You can also use something from the figures if that is easier.