Jumping in planthopper and froghopper insects is propelled by a catapult-like mechanism requiring mechanical storage of energy and its quick release to accelerate the hind legs rapidly. To understand the functional biomechanics involved in these challenging movements, the internal skeleton, tendons and muscles involved were reconstructed in 3-D from confocal scans in unprecedented detail. Energy to power jumping was generated by slow contractions of hind leg depressor muscles and then stored by bending specialised elements of the thoracic skeleton that are composites of the rubbery protein resilin sandwiched between layers of harder cuticle with air-filled tunnels reducing mass. The images showed that the lever arm of the power-producing muscle changed in magnitude during jumping, but at all joint angles would cause depression, suggesting a mechanism by which the stored energy is released. This methodological approach illuminates how miniaturized components interact and function in complex and rapid movements of small animals.DOI:http://dx.doi.org/10.7554/eLife.23824.001 Jumping is one of the fastest and most powerful movements generated by animals. Consequently it places great demands on the nervous system, muscles and the skeleton to ensure that it can be performed successfully and repeatedly. These movements are frequently used to escape from predators so that the cost of failure is high. In insects different jumping mechanisms have evolved independently several times, but all that use the legs for propulsion fall into two broad types. First, contractions of the muscles act directly on long levers. Examples of insects which use this mechanism are bush crickets, the propulsive hind legs of which can be up to 2.6 times the body length . The len−1 in their best jumps  is the rubber-like protein resilin as revealed by staining with acridine orange, but shown in blue , Table 1 mm3 g−1 . In adul mm3 g−1   in the metathorax is very much larger than the equivalent muscles in the other two thoracic segments . The fibThe two levator muscles are much smaller than the whole depressor muscle; the fibres of the larger levator muscle (M132) originate on a lateral, anterior and dorsal coxal process fused with the pleural arch, while the smaller levator muscle (M131) originates on the ventral coxal wall . Their tNymphal froghoppers do not jump and their metathorax lacks the enlarged muscles that power the movements of the hind legs in jumping . The nymLepyronia quadrangularis , which also powers the jump in froghoppers, has a different shape to that in planthoppers . The musThe insertion site for the main medial bundle of fibres that constitute M133c is a surface with edges that curve anteriorly. Close to these insertions and still within the thoracic cavity, the tendon branches into a thinner lateral and anteriorly directed part to which the pinnately arranged fibres of M133b insert along its length. As the combined tendon enters the coxa it tapers gradually to assume a dorso-ventrally flattened shape. Along its medial side is the insertion site of a small part of the muscle (M133a) that originates on the ventral medial wall of the coxa . As in pFirst, the substantially enlarged pleural arches act as energy stores for jumping and provide the necessary rigidity to withstand the forces that are generated. Second, large volumes of resilin are intimately bonded in complex ways with regions of the pleural arches that apparently contain no resilin. These composite structures store and release energy to power repeated jumping. Third, large, paired trochanteral depressor muscles occupy most of the internal volume of the metathorax. The changing lever arms of these muscles suggest a possible mechanism for the release of the stored energy to power a jump. By contrast, the other two pairs of legs which do not contribute propulsion for jumping have smaller pleural arches without resilin and smaller trochanteral depressor muscles.Three dimensional reconstruction of images of the skeletal structures, the muscles and their tendons that generate the rapid propulsive movements of the hind legs, have revealed three features that meet the extraordinary mechanical demands of jumping. In nymphal planthoppers and adult planthoppers and froghoppers, the metathoracic pleural arches are curved bow-like structures with folds and grooves that apparently give increased strength. When the large trochanteral depressor muscles contract slowly before a jump, the pleural arches are bent like archery bows and store elastic energy, but the legs do not move. Sudden release of this stored energy powers the rapid movements of the hind legs in a catapult-like action and drives the insect to a high take-off velocity.The metathoracic pleural arches of all six families of planthoppers analysed had similar common features. A region was identified in the middle of a pleural arch close to where a small laterally placed muscle tendon attaches and two small curved plates also occur. At this point these plates are likely to be subject to the greatest bending forces and might therefore act as additional springs. In planthoppers longitudinal holes are present in the pleural arches and in froghoppers an invagination of the external cuticle forms an air-filled tunnel that extends for some 40% of their length of each arch alongside the cuticular wrapped regions of resilin. These spaces may be a consequence of developmental processes, but functionally they should reduce the mass of the arches and their juxtaposition to the resilin suggests that they may allow easier bending and room for local bulging during energy storage.The protein resilin has previously been detected by its characteristic blue fluorescence under specific wavelengths of UV light , the depIssus coleoptratus . 2. Caliscelidae, Bruchomorpha oculata (Newman 1838). 3. Delphacidae, Stenocranus acutus (Beamer 1946), and Pissonotus sp. 4. Derbidae, Apache degeeri (Kirby 1821). 5. Flatidae, Metcalfa pruinosa (Say 1830) and Flatormenis proxima . 6. Issidae, Thionia bullata (Say 1830), and Issus coleoptratus (Fabricius 1781). Second, the Cercopoidea, (froghoppers) of which there are three families. The froghopper species used here were from the Cercopidae, Lepyronia quadrangularis (Say 1825) and Clastopteridae, Clastoptera obtusa (Say 1825). Both are adept jumpers with similar thoracic anatomies.The Hemipteran insects, suborder Auchenorrhyncha, studied in this paper were collected from similar habitats containing a variety of grasses, wild flowers and shrubs around Ashburn, Virginia, USA and Cambridge, UK in 2014- 2016. They belong to two groups: first, the Fulgoroidea, the planthoppers, of which there are some 20 families; members of six families which are all adept jumpers with similar thoracic anatomies were studied here: 1. Acanaloniidae, For the imaging of cuticle, an insect was anaesthetized on ice and its thorax was isolated in phosphate buffered saline (PBS). The muscles were then digested away with a mixture of hyaluronidase/trypsin at 0.5 mg/ml for 5 hr at 37°C. Resilin of planthoppers and froghoppers was resistant to trypsin, but not to protease K or collagenase. To speed digestion and produce clean cuticle an ultrasonic bath was used towards the end of the process. To render thick samples transparent for imaging it was necessary to match the refractive index of the tissue with that of the mounting medium. The specimens were washed with ethanol and mounted in methyl salicylate . Energy stores prepared in this way were also embedded in 8% agarose and sectioned on Leica Vibratome (VT1000s) at 0.3 mm using the slowest settings for progression of the blade. The sections were mounted in Tris-buffered  50% glycerol.To image cuticle and muscles, the thorax was fixed in 2% paraformaldehyde/PBS for 24–48 hr at room temperature. After washing in PBS containing 1% triton X-100, samples were treated with 0.5 mg/ml hyaluronidase for 2–3 hr at 37°C and briefly sonicated in an ultrasound bath to remove other tissue – mostly fat bodies – in the body cavity. This tissue caused much light scattering so that its removal was essential to obtain clear images of muscles and cuticle. The samples were then stained with Texas Red-X Phalloidin  in PBS with 1% triton X-100, 0.5% DMSO and Escin  at room temperature with agitation for 4–5 days. Long incubation times and the presence of surfactants assured better penetration of phalloidin into the large muscles. In preparations where the 405 nm laser-induced autofuorescence of exoskeletal elements, such as fine tendons in nymphs, was not strong, a chitin-binding dye Calcofluor White  was included in the staining procedure. Acridine orange and Calcofluor White were never used in the same preparation. The samples were then washed in PBS/1% triton and fixed for 4 hr in 2% paraformaldehyde to reduce leaching of bound phalloidin from muscles during the subsequent ethanol dehydration step. To avoid artefacts caused by osmotic shrinkage of soft tissue, samples were gradually dehydrated in glycerol (2–80%) and then ethanol (20% to 100%)  and mounWe attempted to apply the imaging regime/method for visualization of resilin in the exoskeleton of insects as described by Michels and Gorb  but failSerial optical sections were obtained at 2 µm intervals on a Zeiss 710 confocal microscope with a Plan-Apochromat 10x/0.45 NA objective, or at 1 µm with a LD-LCI 25x/0.8 NA objective For the imaging of Acridine Orange-stained cuticle, laser lines of 405 and 488 nm were used. Autofluorescence of chitin was recorded in the 410–465 nm range whereas fluorescence of Acridine Orange was recorded in the 520–580 nm range. The dye exhibits a more red-shifted fluorescence compared to chitin so that collecting emitted light in the 520–580 nm range reduced the chitin autofluorescence component in the registered images. To reduce further the contribution of chitin autofluorescence, we adjusted the offset during recording. Calcofluor White and Texas Red phalloidin-treated samples were imaged using a 405 and 594 nm lasers, respectively. Using these wavelengths for visualization of Calcofluor white-stained exoskeleton and Acridine Orange (AO) -stained resilin creates the possibility of crosstalk. The autofluorescence of resilin would have been included in the visualization; we avoided that by applying Calcofluor White staining only to the nymphs that lack resilin in their energy stores. The settings used to image AO-resilin are appropriate for visualization of exoskeleton structures with large proportions of chitin . The inthttp://fiji.sc/), Icy  and Photoshop (Adobe Systems Inc.). Red/cyan anaglyphs  of the confocal stacks were created using the stereo pair plug-in of ImageJ. The 3-D images should be viewed with glasses in which the left eye has a red filter and the right eye a cyan filter. Images which should be viewed in this way are indicated in the Figures by the symbol of a pair of coloured glasses. 2-D versions of these Figures are also given. In the renderings of confocal images we use blue to indicate resilin (reflecting its characteristic blue fluorescence in UV light), yellow for muscles (it is the only colour showing good contrast in red-cyan anaglyphs) and shades of grey for cuticle.We did not notice substantial differences in the distribution of blue, green and red autofluorescence in cuticular samples. Red autofluorescence was preferentially used for visualization of chitinous elements of the energy stores alone (i.e. when muscles and resilin were not simultaneously imaged), as longer wavelengths are less affected by cuticle pigmentation and thickness, yielding more uniform signal throughout the sample. Autofluorescence of cuticle was excited with a 561 nm laser line and recorded in the 568–683 nm range. The stains and excitation and emission wavelengths used for imaging specimens are listed in The metathoracic muscles involved in jumping are given the same names and numbers  as those used for locusts , becauseIssus coleoptratus were captured by a Photron Fastcam SA3 high speed camera (Photron (Europe), Marlow, Buckinghamshire, UK) at rates of 30 000 frames s–1 and with an exposure time of 0.03 ms. The camera was mounted on a Zeiss Stemi SV6 stereo microscope.Sequential images of jumping movements of  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "3-D reconstruction of energy stores for jumping in planthoppers and froghoppers from confocal scanning microscopy" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This study on planthoppers and froghoppers presents a new functional understanding of jumping that is mechanically insightful, and new, highly detailed anatomy with fantastically rendered images. Using confocal microscopy, physical manipulation, and staining, the authors provide a mechanistic understanding of the interaction of muscle, tendon, and exoskeletal elements, which together conspire to produce synchronous actuation in one of nature's fastest movements.Essential revisions:There are a few key concerns that require revision to the manuscript, summarized below.1) The manuscript suffers a bit by focusing on both new methods (emphasized in the Abstract) and scientific results. The only new method is the staining of resilin with acridine orange, suggesting that the manuscript would benefit most from a stronger focus on the research results. Reviewer #1 has some nice suggestions on how to accomplish such a re-focusing.2) Further details are required to understand the effectiveness of the use of acridine orange for identifying resilin and to justify why this was required compared to autofluorescence (see reviewer #2).3) The more familiar language of levers and mechanical advantage when discussing the findings (see reviewer #3).Reviewer #1:This paper provides an interesting and necessary contribution to current work on the jumping of insects, specifically those who use gears. The image quality, detail, and presentation is stunning.I did find the results difficult to read because the Introduction does not prepare the reader for the topics investigated. Specifically, in the Introduction there are three rough hypotheses which are too vague to actually test. I suggest making predictions here instead. Then use these specific predictions to organize your Results and Discussion. For instance, in the first point you mention the thorax, where power generating muscles are located, must be strong enough to withstand high forces. This suggests you will measure strength, perhaps of the exoskeleton? Instead, a prediction related to the anatomical structure of the thorax would be more appropriate based on your results.It also needs to be clearer why you looked at adults and nymphs and what their differences are. Furthermore, what are the expected differences between planthoppers and froghoppers? Why use multiple species within each? Where are the images of these other species? Are there ecological/behavioral differences that make these comparisons interesting/important?Related to the first two points, certain definitions and details are left out of the Introduction, like resilin, or the ecology of froghoppers, and are included in later sections. Likewise, information on jumping from Burrows would have been useful in the Introduction and would perhaps help formulate predictions .In the Abstract you mention 3D printing of the components, but don't mention this in the manuscript. You are correct that your work could be used for this, but it is not clear how. This paper reads a bit like a methods paper , but it has too much detail with regards to the subject  to be a clear methods paper. Given the data collected, I would prefer this work be focused on structure/function relationships focused around clear predictions.Finally, although the 3D image rendering is impressive, not every reader has the ability to view these with 3D glasses. Can you make 2D images available, perhaps in the supporting information files?Reviewer #2:This manuscript describes the structures involved in the energy storage for jumping of planthoppers and froghoppers in great detail. The 3D reconstructions based on confocal laser scanning microscopy (CLSM) analyses are impressive and provide in fact, as stated by the authors, 'unprecedented detail'. The manuscript is well written and clearly comprehensible, and it adds significant information to the already existing knowledge of the respective structures published in earlier work of Professor Burrows. However, the following aspects and comments have to be considered in the review process:1) The authors stained the exoskeleton structures with large proportions of resilin using the fluorescence dye Acridine Orange. This dye is well-known and typically applied to stain nucleic acids. The finding that Acridine Orange also stains the protein resilin is surprising and very interesting. Unfortunately, the authors only state that 'The distribution of this staining was then confirmed to be the same as that revealed by the characteristic blue fluorescence of resilin when examined with a sharp-edged (1% transmission limits) excitation band of ultraviolet (UV) light.…' but they do not show this. It is absolutely essential that they clearly demonstrate that resilin can be specifically stained with Acridine Orange. For this purpose, the authors definitely have to show the results of precise analyses revealing that the typical resilin autofluorescence and the fluorescence of Acridine Orange are absolutely colocalised in test samples.2) The authors have to convincingly explain why they decided to fluorescently stain resilin with Acridine Orange. This seems to be not very appropriate because the excitation and emission peaks of the Acridine Orange fluorescence are in the ranges of those of an autofluorescence that is typically exhibited by arthropod exoskeleton structures with large proportions of chitin. Accordingly, it is very likely that because of crosstalk the exclusive visualization of the Acridine Orange fluorescence and thereby the specific visualization of the structures with large proportions of resilin within exoskeleton samples  are only possible when a precise emission fingerprinting with detailed λ scans and linear unmixing is performed. Why did the authors not use the resilin autofluorescence? In a relatively large number of earlier studies this autofluorescence has been efficiently used to visualize exoskeleton structures with large proportions of resilin, and it was even demonstrated that a combination of CLSM and an excitation wavelength of 405 nm can be successfully applied to reliably visualize exoskeleton structures with large proportions of resilin .3) The crosstalk problem mentioned above is also present (and even particularly pronounced) in the case of Fluorescent Brightener 28  that was applied by the authors to stain chitin in the exoskeleton and to separately visualize the exoskeleton structures with large proportions of chitin. The excitation and emission properties of the fluorescence of Fluorescent Brightener 28 are comparable to those of the autofluorescence of resilin. Accordingly, it is not easy to separate the different fluorescence signals obtained from Fluorescent Brightener 28 and resilin from each other. Such a separation requires a very precise spectral detection system and a rather precise emission fingerprinting with very detailed λ scans and linear unmixing. In case the particular ZEISS LSM 710 applied by the authors for their study is equipped with a 34-channel QUASAR detection unit, the authors had the opportunity to perform such an emission fingerprinting. Unfortunately, in the manuscript they do not explain at all how the fluorescences were detected. (Which emission wavelength ranges were detected?) Why did the authors decide to apply Fluorescent Brightener 28? They could have used an autofluorescence to visualize the exoskeleton structures with large proportions of chitin. For example, many of such exoskeleton structures exhibit a relatively intense autofluorescence when they are exposed to the light of a 561 nm laser. (Many ZEISS LSM 710 systems are equipped with such a laser.) In this context, another question arises: in the Materials and methods section, the authors mention 'Autofluorescence of cuticle was excited with a 562 nm laser line.' (This must be a mistake because such a laser line is definitely not offered by Carl Zeiss Microscopy. I assume that the authors used a 561 nm laser.), however, this autofluorescence is not mentioned anywhere else in the manuscript. What has happened to the results of this autofluorescence visualization?In conclusion, the authors have to convincingly explain  why they4) In the Discussion section of the manuscript, the authors should clearly highlight which advances their 'new methodological approach' made possible. For this purpose, they should properly discuss the new insights into the energy storage structures important for jumping of planthoppers and froghoppers with respect to the knowledge of the respective structures obtained in earlier studies of Professor Burrows.5) In my opinion, the end of the Discussion section of the manuscript is too abrupt. The authors should add a paragraph with concluding remarks.6) In the Abstract, the authors write 'This new methodological approach can illuminate how miniaturized components interact and function in complex movements of small animals with the potential for 3-D printing of relevant structures.' The potential for 3D printing is not mentioned anywhere else in the manuscript. This topic should be included in the Discussion section of the manuscript.ReferenceMichels J, Gorb SN (2012) Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy. Journal of Microscopy 245, 1-16Reviewer #3:The manuscript entitled "3-D reconstruction of energy stores for jumping in planthoppers and froghoppers from confocal scanning microscopy" describes the detailed structural features that function to store elastic energy and amplify mechanical power in two groups of jumping insects. This work is stunningly beautiful. The level of anatomical detail is indeed unprecedented and set a new standard for future work aiming to derive functional mechanisms from a structural framework. I believe the methods developed here will be of interest to broad audience and likely represents an important contribution to the field. The one potential shortcoming of the paper is that the work is presented in a descriptive way rather than developed hypotheses. In addition, given that the methodology used and developed here is an important aspect of the contribution it may be worth providing the readers with an assessment of the strengths and weaknesses of the approach in the Discussion section. I have also made some stylistic suggestions as well as highlighted a few places where the language could be more precise.eLife. Your revised article has been favorably evaluated by K VijayRaghavan (Senior editor), a Reviewing editor, and three reviewers.Thank you for resubmitting your work entitled "Three dimensional reconstruction of energy stores for jumping in planthoppers and froghoppers from confocal laser scanning microscopy" for further consideration at Please find the reviewers' specific comments below. As you can see, the manuscript has been greatly improved, but there are some remaining issues that need to be addressed before acceptance. For reviewer #1's comments, please try to condense the Discussion further if possible, but more importantly, it would be nice to see a brief conclusion to wrap up the paper. Reviewer #2's comments are more technical in nature and require careful consideration. Reviewer #3 is fully satisfied.Reviewer #1:This manuscript has greatly improved. I appreciate the level of detail the authors used to incorporate reviewer suggestions. In particular, the Introduction now prepares the readers for work discussed in the following sections. I feel the Discussion section is still a bit long and abruptly ends, however I recommend this manuscript for publication. One small note, in subsection “Distribution of resilin in the energy stored of adult planthoppers”, family names are used, rather than the common names used prior to these sentences. It would be helpful to add the common names here so readers not versed in insect taxonomy can follow.Reviewer #2:The authors clearly improved the manuscript. However, there are still a few inconsistencies in the context of the confocal laser scanning microscopy (CLSM) visualizations. These are described below.1) In the first version of the manuscript, the authors wrote “For the imaging of Calcofluor white and Acridine Orange- stained cuticle, laser lines of 405 and 488 nm were used, respectively.” Based on this description, I assumed that the exoskeleton parts with large proportions of chitin had in all cases been visualized by using Calcofluor White M2R and that the exoskeleton parts with large proportions of resilin had in all cases been visualized by using Acridine Orange. And I was already wondering for which visualization the authors had used the autofluorescence that was mentioned in the sentence (but mentioned nowhere else). In the current version of the manuscript, the description is different. The authors now write “For the imaging of Acridine Orange- stained cuticle, laser lines of 405 and 488 nm were used. Autofluorescence of chitin was recorded in the 410-465 nm range;.…” And Calcofluor White M2R is now mentioned (a) Why did the authors use an excitation wavelength of 405 nm and why did they detect fluorescence light with wavelengths of 410-465 nm to visualize exoskeleton parts with large proportions of chitin? The application of quite similar settings (405 nm and 420-480 nm) had been demonstrated to result in the visualization of exoskeleton parts with large proportions of resilin (see Michels and Gorb 2012) but not in the visualization of exoskeleton parts with large proportions of chitin. .) Accordingly, with these settings, the authors did very likely not visualize exoskeleton parts with large proportions of chitin.b) In subsection “Imaging and rendering”, the authors write 'Autofluorescence of cuticle was excited with a 561 nm laser line.' This autofluorescence is also mentioned in the 2) The authors did not really be responsive to my concerns about the large probability of crosstalk having occurred during the CLSM visualizations. They included information about the detected fluorescence wavelengths in the manuscript, and it is now obvious that the applied detection settings had very likely resulted in the occurrence of crosstalk during the CLSM analyses. For example, the Acridine Orange fluorescence, which was used to visualize the exoskeleton structures with large proportions of resilin, was visualized by applying an excitation wavelength of 488 nm and by detecting fluorescence light with wavelengths of 520-580 nm. However, these settings are very appropriate to also visualize an autofluorescence that is typically excited by weakly or non-sclerotised exoskeleton structures with large proportions of chitin (see Michels and Gorb 2012). Accordingly, the authors cannot be sure that they only visualized structures with large proportions of resilin. In the case of the Calcofluor White M2R fluorescence, which was used to visualize the exoskeleton structures with large proportions of chitin, a similar problem exists: this fluorescence was visualized by applying an excitation wavelength of 405 nm and by detecting fluorescence light with wavelengths of 410-570 nm. With these settings, the autofluorescence of resilin was included in the visualization (see above), and the authors cannot be sure that they only visualized structures with large proportions of chitin.It is now of course not possible to get rid of this 'crosstalk problem' anymore. The fluorescences of fluorescence dyes are often much more intense than autofluorescences. In such situations, the proportions of autofluorescences within the fluorescence signal can be minor, and it can happen that the autofluorescences do not negatively affect the analyses. In my opinion, it seems that this was the case in the present study. The results are convincing, and it seems that crosstalk did not play an important role and have a negative effect. However, the authors should mention crosstalk. They should include a statement saying that with the CLSM settings that they applied crosstalk might have occurred but, because of the relatively high intensities of the dye fluorescences compared to those of the autofluorescences, very likely did not negatively influence the results.3) The authors should remove the sub-figures Aiii and Biii from the 4) In the subsection “Imaging and rendering”, the authors should not use the term 'contamination'.ReferenceMichels J, Gorb SN (2012) Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy. Journal of Microscopy 245, 1-16Reviewer #3:The main shortcoming of the original submission was that the text was rather dry and written in a mostly descriptive context based on the visualized morphology. It is worth noting the morphology is some of the most visually stunning, cutting edge, high-resolution imaging I've seen at this scale and therefore is a substantial contribution on its own. However, the paper is now much improved. In particular the new sections in the Discussion elevate the paper beyond descriptive morphology and begin to contextualized the anatomical features in a more mechanical and functional framework. The authors have addressed all of my concerns. I am confident that this paper is a substantial and important contribution.eLife. Your revised article has been favorably evaluated by K VijayRaghavan (Senior editor) and a Reviewing editor.Thank you for resubmitting your work entitled "Three dimensional reconstruction of energy stores for jumping in planthoppers and froghoppers from confocal laser scanning microscopy" for further consideration at The manuscript has been improved sufficiently, but there is one remaining issue that needs to be addressed before acceptance, as described below:Perhaps there was some confusion about reviewer 1's suggestion to provide 2D renderings of 3D images. In your February submission, you suggested that you could provide the images as supplementary material. (Note that this wasn't posed as a question.) Our assumption was that this would be done. Although it is true that 3D glasses are cheap and readily available online, the reality is that some (likely many) readers will not have them on hand, and perhaps won't take the time to order them for a single usage. Thus, providing the images will increase the accessibility of the article. Appendices are allowed by the journal, so it should be straightforward to add an Appendix that includes these images.In particular, please provide 2D versions of each 3D image that hasn't already been provided in the main text. For example, eLife. Your revised article has been favorably evaluated by K VijayRaghavan (Senior editor) and a Reviewing editor.Thank you for resubmitting your work entitled "Three dimensional reconstruction of energy stores for jumping in planthoppers and froghoppers from confocal laser scanning microscopy" for further consideration at The manuscript has been improved sufficiently, but there is one remaining issue that needs to be addressed before acceptance, as described below:The new 2D figures in Appendix 1 look great.  But, all 9 of these figures include 3D glasses icons, which indicates to the reader to use 3D glasses. Was that intended? In the figures in the main body, the glasses icon is only used for 3D-colored images, and not for the 2D images, so the assumption is that this convention holds in the Appendix as well. Essential revisions:There are a few key concerns that require revision to the manuscript, summarized below.1) The manuscript suffers a bit by focusing on both new methods (emphasized in the Abstract) and scientific results. The only new method is the staining of resilin with acridine orange, suggesting that the manuscript would benefit most from a stronger focus on the research results. Reviewer #1 has some nice suggestions on how to accomplish such a re-focusing.We have revised the paper so that it is now focused on the scientific results concerning how energy is stored for jumping and have followed the very helpful comments of reviewer 1. We have removed all comments about new techniques. The Introduction now poses three questions about how the storage devices for jumping store and release energy that are then followed through in the same order in Results and the Discussion.2) Further details are required to understand the effectiveness of the use of acridine orange for identifying resilin and to justify why this was required compared to autofluorescence (see reviewer #2).We have included a supplementary figure that directly addresses this issue. It shows a lack of sufficient dominance of blue fluorescence from resilin over chitin autofluorescence in planthopper and froghopper pleural arches when using the imaging regime suggested by the reviewer and described in Michels and Gorb, 2012. The absence of contrast between those structural elements necessitated the use of staining. The figure also shows side-by-side comparison of wide-field fluorescence micrograph of the resilin autofluorescence in planthopper and froghopper energy stores and the results of Acridine Orange staining imaged with CLSM. We hope the figure convincingly demonstrates colocalization of the characteristic autofluorescence signal and Acridine Orange staining.3) The more familiar language of levers and mechanical advantage when discussing the findings (see reviewer #3).We have changed the language we have used in describing lever arms and mechanical advantages to that suggested by reviewer 3 throughout the paper. We have consulted a mechanical engineer to ensure that our descriptions are correct, and have checked carefully for consistency.Reviewer #1:This paper provides an interesting and necessary contribution to current work on the jumping of insects, specifically those who use gears. The image quality, detail, and presentation is stunning.I did find the results difficult to read because the Introduction does not prepare the reader for the topics investigated. Specifically, in the Introduction there are three rough hypotheses which are too vague to actually test. I suggest making predictions here instead. Then use these specific predictions to organize your Results and Discussion. For instance, in the first point you mention the thorax, where power generating muscles are located, must be strong enough to withstand high forces. This suggests you will measure strength, perhaps of the exoskeleton? Instead, a prediction related to the anatomical structure of the thorax would be more appropriate based on your results.It also needs to be clearer why you looked at adults and nymphs and what their differences are. Furthermore, what are the expected differences between planthoppers and froghoppers? Why use multiple species within each? Where are the images of these other species? Are there ecological/behavioral differences that make these comparisons interesting/important?The biology that we have added to the Introduction now explains that nymphal planthoppers but not nymphal froghoppers are adept at jumping like the adults. We have used different species from different families to demonstrate the generalities of the structures and mechanisms within the two groups. The species illustrated are named in each of the figures. The insects analysed were found in similar habitats (but we can’t assume that this is true for the many species that belong to these two groups) and show broadly similar behaviour – particularly as far as jumping – but detailed knowledge is not available in the literature.Related to the first two points, certain definitions and details are left out of the Introduction, like resilin, or the ecology of froghoppers, and are included in later sections. Likewise, information on jumping from Burrows would have been useful in the Introduction and would perhaps help formulate predictions .We have moved all these points to the Introduction and have expanded some to give the reader a broader background to this study.In the Abstract you mention 3D printing of the components, but don't mention this in the manuscript. You are correct that your work could be used for this, but it is not clear how.We have deleted this superfluous mention of 3D printing.This paper reads a bit like a methods paper , but it has too much detail with regards to the subject  to be a clear methods paper. Given the data collected, I would prefer this work be focused on structure/function relationships focused around clear predictions.We have refocused the paper so that we hope it no longer reads a bit like a methods paper. It was always our intention that the focus was on answering specific questions of how the energy for jumping was stored. We would hope that readers will be encouraged by the power of the methodology we have used when applied to functional analyses of movement.Finally, although the 3D image rendering is impressive, not every reader has the ability to view these with 3D glasses. Can you make 2D images available, perhaps in the supporting information files?We have agonized over this problem  but did not know how to resolve it. Your suggestion is very helpful and if the journal would allow us to do this, we would be happy to duplicate the 3D figures in 2D for the supplementary material. But there would of course be a loss of some information that only the glasses could bring out.Reviewer #2:This manuscript describes the structures involved in the energy storage for jumping of planthoppers and froghoppers in great detail. The 3D reconstructions based on confocal laser scanning microscopy (CLSM) analyses are impressive and provide in fact, as stated by the authors, 'unprecedented detail'. The manuscript is well written and clearly comprehensible, and it adds significant information to the already existing knowledge of the respective structures published in earlier work of Professor Burrows. However, the following aspects and comments have to be considered in the review process:1) The authors stained the exoskeleton structures with large proportions of resilin using the fluorescence dye Acridine Orange. This dye is well-known and typically applied to stain nucleic acids. The finding that Acridine Orange also stains the protein resilin is surprising and very interesting. Unfortunately, the authors only state that 'The distribution of this staining was then confirmed to be the same as that revealed by the characteristic blue fluorescence of resilin when examined with a sharp-edged (1% transmission limits) excitation band of ultraviolet (UV) light.…' but they do not show this. It is absolutely essential that they clearly demonstrate that resilin can be specifically stained with Acridine Orange. For this purpose, the authors definitely have to show the results of precise analyses revealing that the typical resilin autofluorescence and the fluorescence of Acridine Orange are absolutely colocalised in test samples.We have included a supplementary figure showing side-by-side comparison of wide-field fluorescence micrograph of the resilin autofluorescence in planthopper and froghopper pleural arches and the results of Acridine Orange staining imaged with CLSM. In this comparison the natural fluorescence of the resilin matches exactly with the staining with AO. Please note that the differences in the distribution of resilin in planthoppers and froghoppers is also visible in both the autofluorescence images and the AO staining. We hope the figure convincingly demonstrate colocalization of the characteristic autofluorescence signal and Acridine Orange staining.2) The authors have to convincingly explain why they decided to fluorescently stain resilin with Acridine Orange. This seems to be not very appropriate because the excitation and emission peaks of the Acridine Orange fluorescence are in the ranges of those of an autofluorescence that is typically exhibited by arthropod exoskeleton structures with large proportions of chitin. Accordingly, it is very likely that because of crosstalk the exclusive visualization of the Acridine Orange fluorescence and thereby the specific visualization of the structures with large proportions of resilin within exoskeleton samples  are only possible when a precise emission fingerprinting with detailed λ scans and linear unmixing is performed. Why did the authors not use the resilin autofluorescence? In a relatively large number of earlier studies this autofluorescence has been efficiently used to visualize exoskeleton structures with large proportions of resilin, and it was even demonstrated that a combination of CLSM and an excitation wavelength of 405 nm can be successfully applied to reliably visualize exoskeleton structures with large proportions of resilin .We have indeed tried to use the imaging procedure described by Michels and Gorb in their beautiful 2012 work, but we were unable to observe a pronounced dominance of blue fluorescence from resilin. We therefore did not obtain satisfactory contrast between highly sclerotized cuticle and resilin in these energy stores. We included the examples of our attempts in the supplementary figure (panel ii). Instead we found out that resilin stains very strongly with relatively dilute solution of Acridine Orange and the emitted fluorescence is overwhelmingly stronger than that of the surrounding chitinous structures. We now show in 3) The crosstalk problem mentioned above is also present (and even particularly pronounced) in the case of Fluorescent Brightener 28  that was applied by the authors to stain chitin in the exoskeleton and to separately visualize the exoskeleton structures with large proportions of chitin. The excitation and emission properties of the fluorescence of Fluorescent Brightener 28 are comparable to those of the autofluorescence of resilin. Accordingly, it is not easy to separate the different fluorescence signals obtained from Fluorescent Brightener 28 and resilin from each other. Such a separation requires a very precise spectral detection system and a rather precise emission fingerprinting with very detailed λ scans and linear unmixing. In case the particular ZEISS LSM 710 applied by the authors for their study is equipped with a 34-channel QUASAR detection unit, the authors had the opportunity to perform such an emission fingerprinting. Unfortunately, in the manuscript they do not explain at all how the fluorescences were detected. (Which emission wavelength ranges were detected?) Why did the authors decide to apply Fluorescent Brightener 28? They could have used an autofluorescence to visualize the exoskeleton structures with large proportions of chitin. For example, many of such exoskeleton structures exhibit a relatively intense autofluorescence when they are exposed to the light of a 561 nm laser. (Many ZEISS LSM 710 systems are equipped with such a laser.) In this context, another question arises: in the Materials and methods section, the authors mention 'Autofluorescence of cuticle was excited with a 562 nm laser line.' (This must be a mistake because such a laser line is definitely not offered by Carl Zeiss Microscopy. I assume that the authors used a 561 nm laser.), however, this autofluorescence is not mentioned anywhere else in the manuscript. What has happened to the results of this autofluorescence visualization?In conclusion, the authors have to convincingly explain  why theyWe were not able to obtain satisfactory separation of the resilin and chitin autofluorescence in adult plant- and froghopper energy stores. The autofluorescence of their highly sclerotised exoskeleton of the adults was intense enough when either excitatory light of 405, 488 or 561 nm was used; when imaging chitinous elements of the pleural arches alone we did use 561 nm laser line (we have corrected our mislabeling of this wavelength). In contrast to the adults, 405 nm laser-induced autofuorescence of exoskeletal elements such as fine tendons in nymphs was not strong enough and we decided to augment it with Calcofluor. The blue-green fluorescence of Clacofluor does not bleed into Texas Red Phalloidin channel and we successfully used this combination of dyes for simultaneous 3-D imaging of exoskeleton and muscles. We did not combine Acridine Orange and Calcofluor, opting instead for 405 nm- excited autofluorescence of chitin (see above). To make our methodology clear we have added a table in the Materials and methods section, listing the stains and wavelengths used to image different tissues.4) In the Discussion section of the manuscript, the authors should clearly highlight which advances their 'new methodological approach' made possible. For this purpose, they should properly discuss the new insights into the energy storage structures important for jumping of planthoppers and froghoppers with respect to the knowledge of the respective structures obtained in earlier studies of Professor Burrows.We have now deleted all suggestions that this is a new methodological approach and instead focus on the answers that we pose in the Introduction and how these relate to the problems of jumping with such speed and power.5) In my opinion, the end of the Discussion section of the manuscript is too abrupt. The authors should add a paragraph with concluding remarks.We have rewritten the last section of the Discussion to avoid this abrupt ending. Please see our response to reviewer 2 comment.6) In the Abstract, the authors write 'This new methodological approach can illuminate how miniaturized components interact and function in complex movements of small animals with the potential for 3-D printing of relevant structures.' The potential for 3D printing is not mentioned anywhere else in the manuscript. This topic should be included in the Discussion section of the manuscript.We have deleted the mention of 3D printing.ReferenceMichels J, Gorb SN (2012) Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy. Journal of Microscopy 245, 1-16We have added this reference.Reviewer #1:This manuscript has greatly improved. I appreciate the level of detail the authors used to incorporate reviewer suggestions. In particular, the Introduction now prepares the readers for work discussed in the following sections. I feel the Discussion section is still a bit long and abruptly ends, however I recommend this manuscript for publication. One small note, in subsection “Distribution of resilin in the energy stored of adult planthoppers”, family names are used, rather than the common names used prior to these sentences. It would be helpful to add the common names here so readers not versed in insect taxonomy can follow.Our problem is that there are no common names for these two families or their members. A compromise that we have adopted is to call them Derbid planthoppers and Acanaloniid planthoppers which thus includes the family name and the general group name of planthoppers.Reviewer #2:The authors clearly improved the manuscript. However, there are still a few inconsistencies in the context of the confocal laser scanning microscopy (CLSM) visualizations. These are described below.1) In the first version of the manuscript, the authors wrote “For the imaging of Calcofluor white and Acridine Orange- stained cuticle, laser lines of 405 and 488 nm were used, respectively.” Based on this description, I assumed that the exoskeleton parts with large proportions of chitin had in all cases been visualized by using Calcofluor White M2R and that the exoskeleton parts with large proportions of resilin had in all cases been visualized by using Acridine Orange. And I was already wondering for which visualization the authors had used the autofluorescence that was mentioned in the sentence (but mentioned nowhere else). In the current version of the manuscript, the description is different. The authors now write “For the imaging of Acridine Orange- stained cuticle, laser lines of 405 and 488 nm were used. Autofluorescence of chitin was recorded in the 410-465 nm range;.…” And Calcofluor White M2R is now mentioned  We have reformatted 2) We say “In preparations where the 405 nm laser-induced autofuorescence of exoskeletal elements, such as fine tendons in nymphs, was not strong, a chitin-binding dye Calcofluor White  was included in the staining procedure. Acridine orange and Calcofluor White were never used in the same preparation.”3) We say “Autofluorescence of chitin was recorded in the 410-465 nm range whereas fluorescence of Acridine Orange was recorded in the 520-580 nm range.”a) Why did the authors use an excitation wavelength of 405 nm and why did they detect fluorescence light with wavelengths of 410-465 nm to visualize exoskeleton parts with large proportions of chitin? The application of quite similar settings (405 nm and 420-480 nm) had been demonstrated to result in the visualization of exoskeleton parts with large proportions of resilin (see Michels and Gorb 2012) but not in the visualization of exoskeleton parts with large proportions of chitin. .) Accordingly, with these settings, the authors did very likely not visualize exoskeleton parts with large proportions of chitin.We did not notice substantial differences in the distribution of blue, green and red autofluorescence in cuticular samples. Similarly, the boundaries of the detected wavelengths did not have a marked effect on visualization of the energy stores.We now say: “We did not notice substantial differences in the distribution of blue, green and red autofluorescence in cuticular samples.”b) In subsection “Imaging and rendering”, the authors write 'Autofluorescence of cuticle was excited with a 561 nm laser line.' This autofluorescence is also mentioned in the We added a sentence clarifying that red autofluorescence was used for visualization of chitinous elements of energy stores alone (i.e. when muscles and resilin were not simultaneously imaged).“Red autoflorescence was preferentially used for visualization of chitinous elements of the energy stores alone (i.e. when muscles and resilin were not simultaneously imaged), as longer wavelengths are less affected by cuticle pigmentation and thickness, yielding a more uniform signal throughout the sample. Autofluorescence of cuticle was excited with a 561 nm laser line and recorded in the 568-683 nm range.”We also included the information about using red autofluorescence in relevant figure legends.2) The authors did not really be responsive to my concerns about the large probability of crosstalk having occurred during the CLSM visualizations. They included information about the detected fluorescence wavelengths in the manuscript, and it is now obvious that the applied detection settings had very likely resulted in the occurrence of crosstalk during the CLSM analyses. For example, the Acridine Orange fluorescence, which was used to visualize the exoskeleton structures with large proportions of resilin, was visualized by applying an excitation wavelength of 488 nm and by detecting fluorescence light with wavelengths of 520-580 nm. However, these settings are very appropriate to also visualize an autofluorescence that is typically excited by weakly or non-sclerotised exoskeleton structures with large proportions of chitin (see Michels and Gorb 2012). Accordingly, the authors cannot be sure that they only visualized structures with large proportions of resilin. In the case of the Calcofluor White M2R fluorescence, which was used to visualize the exoskeleton structures with large proportions of chitin, a similar problem exists: this fluorescence was visualized by applying an excitation wavelength of 405 nm and by detecting fluorescence light with wavelengths of 410-570 nm. With these settings, the autofluorescence of resilin was included in the visualization (see above), and the authors cannot be sure that they only visualized structures with large proportions of chitin.It is now of course not possible to get rid of this 'crosstalk problem' anymore. The fluorescences of fluorescence dyes are often much more intense than autofluorescences. In such situations, the proportions of autofluorescences within the fluorescence signal can be minor, and it can happen that the autofluorescences do not negatively affect the analyses. In my opinion, it seems that this was the case in the present study. The results are convincing, and it seems that crosstalk did not play an important role and have a negative effect. However, the authors should mention crosstalk. They should include a statement saying that with the CLSM settings that they applied crosstalk might have occurred but, because of the relatively high intensities of the dye fluorescences compared to those of the autofluorescences, very likely did not negatively influence the results.We agree that some crosstalk may occur in visualizing the cuticular elements of the energy stores. We already described our approach to reducing the chitin autofluorescence component during visualization of Acridine Orange-stained resilin. We now added the following text for further clarification:“Using these wavelengths for visualization of calcofluor white-stained exoskeleton and Acridine Orange (AO) -stained resilin creates the possibility of crosstalk. […]The intensity of AO fluorescence is much stronger than autofluorescence of chitin, however, so that the crosstalk did not play an important role in our analysis of the distribution of resilin and did not impact the results.”Our conclusion is thus in agreement with that of the reviewer “because of the relatively high intensities of the dye fluorescences compared to those of the autofluorescences, very likely did not negatively influence the results.”3) The authors should remove the sub-figures Aiii and Biii from the 'We have removed panels Aiii and Biii from the supplementary figure and all references to them in the text.4) In the subsection “Imaging and rendering”, the authors should not use the term 'contamination'.We replaced “contamination” with “contribution”.


To identify a typology of heavier drinking styles in Great Britain and to identify socio-demographic trends in the typology over the period 1978–2010.We applied multiple correspondence analysis and agglomerative hierarchical clustering to beverage-specific quantity–frequency measures of alcohol consumption in the repeated cross-sectional General Lifestyle Survey of Great Britain, 1978–2010. The cluster analysis focuses on the 60,043 adult respondents over this period reporting average drinking levels above the UK Government guidelines. We projected sex, age, income, education, socio-economic status and tobacco consumption variables onto the clusters to inspect socio-demographic trends in heavier drinking.We identified four stable clusters of heavier drinking: (a) high volume beer; (b) beer and spirit combination; (c) all beverage and (d) wine and spirit only. The socio-demographic characteristics of the clusters were distinct from both each other and the general population. However, all clusters had higher median incomes and higher smoking rates than the population. Increases in the prevalence of heavier drinking were driven by a 5-fold increase in the contribution of the female-dominated, wine and spirit only cluster.Recent changes in per capita alcohol consumption in Great Britain occurred within the context of a stable typology of heavier drinking styles and shifting socio-demographics. Identifying these trends is essential to better understand how drinking cultures develop over time and where potentially problematic drinking styles may emerge. Our findings suggest that careful attention to patterns and cultures of consumption is more important than relying on headline consumption data, for both understanding drinking behaviours and targeting interventions.This analysis of alcohol consumption survey data identifies four styles of heavier drinking in Great Britain, which remain unchanged over the period 1978–2010. The socio-demographic characteristics of the drinking styles are distinct from both each other and the general population, with increased participation of female and older drinkers over time. The question of how and why population-level patterns of alcohol consumption change is of fundamental importance to alcohol research . This deWhile some recent studies have offered conceptual models for making sense of changes within national drinking cultures . ConventWhilst we might conceive of alcohol consumption as being composed of a rich tapestry of drinking occasions  since, together with income, it is used in health equality analysis—specifically, the ‘alcohol harm paradox’ whereby deprived people are disproportionately affected by alcohol harms in relation to their levels of drinking , which is a repeated cross-sectional survey of UK households that collected data on alcohol consumption (using a beverage-specific quantity–frequency (QF) method) between 1978 and 2010 . Averagedrinking  and may To focus on the subpopulation drinking at levels above the UK guidelines during the period of the study, we analyse individuals reporting drinking at ‘increasing risk’  and ‘higher risk’  levels. One unit is equivalent to 10 ml or 8 g ethanol. Collectively, we refer to this subpopulation of individuals as ‘heavier drinkers’.We use a two-step procedure to identify drinking clusters for each year of GLF data. Firstly, we reduce the dimensionality of the QF data and, secondly, we use formal clustering methods to identify groups in this lower dimensional space. The underpinning methodology follows previous work by We use the method of multiple correspondence analysis (MCA) to perform the dimensionality reduction . MCA is Every respondent in the data can be projected on to the identified lower dimensional Euclidean representation to produce a so-called cloud of individuals . To idenThe population characteristics of the clusters are analysed by treating demographics as supplementary variables—such variables are simply treated as projections, and are not used to produce the clusters themselves. Post-processing of results has been performed in Matlab R2014a.m1, m2 and m3) of the MCA are 0.58, 0.18 and 0.09. These axes produce a cumulative rate of 0.85, which meets the threshold for selection of a reduced-dimension Euclidean space. Next we consider the contributions of the QF categories to each principal axis, and the direction of each of these contributions. In Table m1 distinguishes between (a) bingeing beer consumption (>8 units per occasion) combined with abstention from other beverage types and (b) the consumption of wine (2–4 units most days) and fortified wine (up to 2 units possibly weekly). Axis m2 distinguishes between (c) frequent wine consumption combined with abstention from other beverages and (d) frequent, above daily guidelines beer consumption (4–8 units some days of the week). Axis m3 distinguishes between (e) weekly spirit consumption (at above daily guidelines or binge levels) combined with abstention from beer and (f) frequent wine consumption combined with occasional moderate beer drinking.MCA is applied to QF data for 60,043 heavier drinkers  over the period 1978–2010. The modified variance rates for the first three principal axes  has a centroid indicating a strong preference for binges on beer, with weaker preferences for wine drinking. The centroid of the fourth cluster  indicates a strong preference for frequent wine consumption combined with a moderate preference for weekly spirit drinking, both to the complete exclusion of beer. We consider these clusters as representing two drinking styles: ‘high volume beer’ drinking and ‘wine and spirit only’ drinking, respectively.As suggested by the median weekly consumption levels, the majority of each cluster comprised increasing risk, as opposed to higher risk, drinkers. For example, in 1992, the relative prevalence of increasing risk consumption ranged from 77% for the ‘beer and spirit combination’ cluster to 87% for the ‘wine and spirit only’ cluster.Figure As seen in Figure Drinker dynamics for gender, age, income, education, SES and smoking, arising from analysis of the supplementary variables, are shown in Figure From Figure As shown in Figure As shown in Figure Results showing the proportion of individuals in each cluster who received a higher education (HE) are shown in Figure Figure From Figure The increasing prevalence of female heavier drinking seen in this study reinforces the conclusions of existing studies of population drinking dynamics—all of which have used GLF data. Our analysis adds more detail to this picture of female drinking dynamics. The increasing prevalence of heavier drinking in the 1930–1940s birth cohorts was strongly attracted to the existing female-dominant drinking style of ‘wine and spirit only’. This is evident from the ageing of this cluster during the early to mid-1990s when, according This analysis brings us new perspectives on developments in British drinking cultures since the 1980s. Over this period, both the legislative framework and market structures around alcohol have changed dramatically . This shThe long increase in per capita consumption between 1980 and the mid-2000s (an increase which began in the early 1970s) was driven, primarily, by an expanding wine market but also by higher strength beers and the development of new products, including—but only to a limited extent—ready-to-drink (RTDs) mixers, also known as ‘alcopops’. Until now, however, our understanding of the extent to which these trends can be attributed to high-consuming subgroups has been limited. This has meant that debates on the relationship between population trends and harmful consumption have, outside of the specialist research community at least, often relied on somewhat crude assumptions regarding overall proportions of moderate, hazardous and harmful drinking in the population. At the same time, much media reporting have reinforced the incorrect assumption that consumption increases are isolated primarily among young people consuming RTDs, lager and spirits . By contMCA and clustering are exploratory data analysis methods that lack the formal hypothesis testing instruments associated with more conventional analysis methods such as APC and time series. Therefore, it is important to avoid making too strong an interpretation of our findings. However, we stress that the absence of goodness-of-fit metrics and significance tests needs to be balanced against the advantages of MCA and clustering for identifying nonlinear relationships in the data—a key concern for alcohol research. We have been able to identify dichotomies and dynamics . However, it is possible that higher order moments and richer categorical demographic indicators might modify some of the conclusions. Further limitations relate to issues with the GLF itself. Since the survey is not longitudinal, we cannot track the relative stability of individuals within clusters over time. In the long-run data, the QF questions are separated by beverage, so drinking occasions that comprise beverage combinations cannot be identified, and the diversity of drinking occasions that can be identified is also limited (It is important to recognize that GLF does not capture all of the population subgroups that might include heavier drinkers; people who are homeless or are resident in institutions, care homes or prisons are not sampled, whilst students and dependent drinkers are under-sampled  suggestsDrinking typologies tend to be conceived in the literature as classes of developmental trajectories for individuals, typically identified from cohort studies; see Supplementary DataClick here for additional data file.


The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21’s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.DOI:http://dx.doi.org/10.7554/eLife.18020.001 Cancer develops when cells in the body mutate in ways that allow them to rapidly grow and divide. To protect cells from becoming cancerous, various molecules act like guardians to prevent cells from dividing when their DNA is damaged, or if they are short of energy. Other guardian molecules monitor the DNA copying process to ensure that the newly-made DNA is as identical as possible to the original DNA template.A protein called p21 belongs to the first group of guardian molecules: DNA damage triggers the production of p21, which prevents the cell from copying its DNA. This role relies on a section of the protein called the CDK binding domain. Cells that have already started to copy their genetic material also have low levels of p21.Mansilla et al. used human cells to investigate whether p21 is also involved in the process of copying DNA. The experiments show that the low levels of p21 act to increase the speed at which the DNA is copied. This activity helps to ensure that all of the cell’s DNA is copied within the time available, including sections of DNA that are harder to copy because they are more fragile and prone to damage. This newly identified role does not involve the CDK binding domain, but instead requires a different section of the p21 protein known as the PCNA interacting region.Mansilla et al. propose that p21 plays a dual role in protecting us from developing cancer. The PCNA interacting region is also found in other proteins that are involved in copying DNA. Therefore, a future challenge is to find out how these proteins interact with each other to ensure that cells accurately copy their DNA in a timely fashion.DOI:http://dx.doi.org/10.7554/eLife.18020.002 CDKN1A and p21Cip1/Waf1), is a member of the family of cyclin-dependent kinase (CDK) inhibitors (CKIs) which has long been known for its ability to consolidate the G1 and G2 arrest after DNA damage caused by genotoxic agents, such as γ irradiation (γ IR)  . As p21 n (γ IR) . Such obn (γ IR) .However, p21 levels in cycling cells are not null. Albeit p21 does not efficiently inhibit CDK activity in cycling cells  it couldAs DNA polymerases (pols) also bind the IDCL of PCNA through PIR or PIP (PCNA interacting protein) boxes, p21 should be capable of negatively regulating all PCNA-dependent DNA synthesis processes in cells . In factCdt2 E3 ligase at replisomes  and Methyl methane sulfonate (MMS) treatments . We haveplisomes . The CRLted PCNA . The listed PCNA . In concWhile the above mentioned reports demonstrate the relevance of disrupting p21-PCNA interaction in cells, no report has ever addressed the relevance of the PCNA-p21 complex in cells. Here we report that endogenous p21 localizes at replication factories through PCNA binding, thereby avoiding DNA polymerase κ (Pol κ) to be recruited at replication factories. Surprisingly, in contradiction with its function as a negative regulator of CDKs, p21 facilitates S phase progression; that is p21 promotes nascent DNA elongation.The DNA replication defects caused by p21-depletion caused accumulation of replication stress markers, such as γH2AX and 53BP1, instability of common fragile sites and micronuclei (MN) accumulation. Interestingly, all the replication defects observed in p21-depleted cells were eliminated when Pol κ was depleted, and were also complemented by a p21 mutant with an intact PCNA binding domain and a disrupted CDK binding site. Collectively, our data demonstrate that, although expressed at low levels in S phase, p21 fine-tunes the dynamics of DNA replication by regulating Pol κ loading to replisomes. Therefore, while the CDKs/p21 interaction is crucial to the cellular response to DNA damage, the PCNA/p21 interaction prevents the accumulation of DNA-damage independent genomic instability in unstressed cycling cells.PIPMut*, bearing an intact or a disrupted PIR, respectively , followed by a 30-min pulse of IdU (Iododeoxyuridine). After denaturalization, stretching and labelling with specific antibodies, DNA track lengths were quantified. Surprisingly, p21 loss resulted in a reduced track length suggesting that p21 is required for a proper elongation of the replication forks  in binucleated cells that have finished karyocinesis . NotablyBesides p21, novel negative regulators of alt DNA pols have been recently identified . One of T. Huang and colleagues have previously reported that MN accumulation induced by USP1 depletion depends on Pol κ . TherefoWhile MN accumulation was evident when p21 was knocked down, this was not observed after simultaneous depletion of Pol κ and p21 . CFS insThe recruitment of the alternative DNA polymerase pol eta (Pol η) to replication factories was also stimulated in the absence of p21 . Howeverbona-fide marker of G2/M transition  human foreskin fibroblast (HFF) and (b) mesenchyimal stem cells isolated from umbilical cord (MSC). As shown in We describe a biologically relevant contribution of the interaction of p21 and PCNA in cells. We show that such interaction improves DNA replication dynamics since it is required to ensure the best rate of nascent DNA elongation. By promoting accurate DNA polymerase choice at the replisome, p21 acts as a tumor suppressor chronically at each duplication cycle, in the absence of exogenous sources of DNA damage.An important implication of our findings is that the idea that p21 acts exclusively as a negative regulator of the cell cycle through the inhibition of cycling kinases  is simplPrevious work indicates that p21 can displace alt DNA pols from replicating DNA. First, Z. Livneh and colleagues showed that p21 impairs TLS events on transfected plasmids . Second,Cdt2 might rapidly allow Pol κ binding to others. Such p21 function could be executed either by promoting the dissociation of Pol κ from replisomes or by preventing its recruitment to DNA regions that must be necessarily copied by replicative DNA pols (such as for example B-regions in the exonic DNA).There is a tight cross-regulation of PIP box-containing proteins at replication forks which is not yet completely understood. On the one hand, it has been demonstrated that proteins with strong PIP boxes such as p21 can remove alt pols such as Pol κ from replication factories (this report and ). On theCDT2 may maintain the most efficient DNA elongation speed by allowing the rapid and dynamic exchange of replicative DNA pols for Pol κ. In fact, a minor shift in the timing of CLR4CDT2-dependent p21 degradation is sufficient to accumulate cells in S phase  aggravate the defect  or (b) not alter such defect   events. While such p21 function has been linked to CDK inhibition  . As our hibition , it is shttp://iclac.org/databases/cross-contaminations/). Samples were grown in Dulbecco’s modified Eagle’s medium  with 10% fetal calf serum in a 5% CO2 atmosphere. Samples are routinely tested for mycoplasma by PCR every two weeks.All cell lines were received from reliable sources, either public repositories or directly, from the laboratory that generated them. They were never frozen at passage higher than eight. U2OS (source: ATCC), and HCT116 p21+/+ and HCT116 p21−/−  were received directly from ATCC and Johns Hopkins University respectively. Samples were minimally amplified and frozen after the reception and were used for limited passages after thawing of those primary stocks. We have verified the identity of the cell lines in terms of the pathways which are key for this study. We have verified the p53 and p21 status by RT-PCR. The three cell lines are positive for p53 and only U2OS and HCT 116 p21+/+ were positive for p21, while HCT 116 p21−/− was not. None of the used cell lines were in the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee  updated Umbilical cord mesenchymal stem cells (UC-MSC) were isolated from Wharton jelly tissues. Small pieces of the umbilical cord, excluding the major vessels, were layered onto plastic culture plates and cultured in D-MEM supplemented with 10% SFB and penicillin-streptomycin. After 14 days, cells started to emerge from the tissue pieces. When plates were almost confluent, umbilical cord pieces were removed and cells trypsinized and frozen. Multilineage differentiation potential of UC-MSC was assessed using StemPro Adipogenesis, Osteogenesis or Chondrogenesis Kit  as per instruction of the manufacturer. Human Foreskin Fibroblasts (HFF) were obtained under informed consent from his parents from the foreskin of a three-year old boy undergoing a scheduled surgery. HFF were obtained using a similar protocol to the one used with UC-MSC. Both cell lines were grown in DMEM supplemented with 10% FBS and antibiotics up to a 15 passage. All isolations of primary cells were approved by FLENI Ethic Committee after reviewing the research protocol .The HFF (Human Foreskin Fibroblasts) were analyzed by karyotyping to assess genomic integrity after 10 passages. Experiments were performed using cells up to passage number 15 to prevent the occurrence of abnormalities. In addition, cells surface markers are periodically analized by flow citometry. MSC line were used up to passage number 10 to prevent genomic aberrations and lose of stem cells´ properties. MSC surface marker expression profile and their differentiation potential were periodically analized by flow citometry and by chondregic, adipogenic and osteogenic differentiation, respectively. MSC immunomodulatory properties were also assessed by CFSE lymphocyte proliferation assay. All cell lines were weekly analyzed for mycoplasm contamination by PCR. Transfections of siRNAs were performed using Lipofectamine RNAiMAX (Thermofisher) and Jet Prime  was used when delivering both siRNAs and plasmid expression vectors.GFP-Pol η and GFP-Pol κ were gifts from Dr. A Lehmann . GFP-PCNsiRNA p21 3'UTR:GGAACAAGGAGUCAGACAU;siRNAUSP1:UCGGCAAUACUUGCUAUCUUA ;siRNA Pol κ1: AAGAUUAUGAAGCCCAUCCAA ;siRNA Pol κ2: AACCUCUAGAAAUGUCUCAUA siRNA Pol η: CUGGUUGUGAGCAUUCGUGUA .PIPMut*  which disrupt the CDK2/p21 interaction  and generated the p21eraction ) Both theraction . Primerseraction  and botheraction .PIPMut* were cloned into the lentiviral transfer plasmid pLenti CMV/TO Puro  using BamHI and XbaI. Packaging and envelope 2nd generation vectors used were psPAX2 and PMD2.G respectively. Lentiviral particles were obtained by transfecting with Jet prime Hek293T cells in 60 mm dishes in a 5:5:1 ratio (pLenti: psPAX2:PMD2). 48 hr after transfection lentiviral particles were collected from the supernatant of Hek293T cells, centrifuged, filtered with a 0.45 μm filter and stored at −80°C. Lentiviral preparations were slowly thawed and used to infect U2OS cells in 24/12 well dishes in the presence of 8 µg/ml of polybrene. Infection efficiency was approximately 90% /2% sucrose and permeablized with 0.1% Triton X-100 in phosphate buffered saline (PBS) as described previously . For theFor Western blot analysis, samples were lysed in Laemmli buffer. Antibodies used were anti-p21  anti-polη , anti-KU70 , and anti-GFP (Santa Cruz Biotechnology). Secondary antibodies (Sigma) and ECL detection  were used according to the manufacturers’ instructions. Western blot images were taken with Image QuantLAS4000 , which allows capture and quantification of images within a linear range. These images were then quantified with the ImageJ software when indicated.Pennsylvania), rat anti-BrdU  to detect CldU and donkey antirat Alexa 488 secondary antibody (Invitrogen). Slides were mounted with Mowiol 488 , and DNA fibers were visualized using a Zeiss Axioplanconfocal microscope. Images were analysed using Zeiss LSM Image Browser software and Image J software. Each data set is derived from measurement of 85–100 fibers.DNA fibers were analysed using a protocol previously used by us  with a mChromatin immunoprecipitations were performed as described in . U2OS ceU2OS cells were seeded onto 22 mm x 22 mm coverslips in 6 well plates. 24 hr later cells were pre-extracted with ice cold CSK buffer  for 2 min and fixed with PFA 4% for 10 min. After pre-extraction and fixation, interaction between endogenous p21 and PCNA was detected following manufacturer’s instructions Duolink In Situ – Fluorescence (SIGMA). Briefly PLA’s principle is based on detecting proteins in close proximity (30–40 nm) by using different species of primary antibodies. A pair of oligonucleotide (PLA probes) detect the primary antibodies bound the proteins of interest and generates a signal only when the two PLA probes have bound in close proximity, meaning that the samples are localized in close proximity. The signal from each detected pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) and assigned to a specific subcellular location based on microscopic images. Images were obtained with a Zeiss Axioplan confocal microscope and PLA spots were counted using Image J software, cells were counted as positive when more than 3 PLA spots were detected. When analyzing pol κ/PCNA interaction a pol κ 'home made' monoclonal antibody from mice purified by Biotem (clone n°16A7-3A11) and PCNA rabbit polyclonal (Abcam ref ab18197) were used. For the quantification of number of foci per nuclei the Cell Profiler program was used to analyze more than 1000 nuclei.After transfection with the indicated siRNAs, cells were lysed and total RNA was extracted using TRIzol Reagent (Invitrogen). Total RNA (1 μg) was used as a template for cDNA synthesis utilizing an ImProm-II Reverse Transcription System (Promega) and oligo-dT as a primer. Quantitative real-time PCR was performed using the MX3005P quantitative PCR instrument (Stratagene) with Taq DNA poly-merase (Invitrogen) and SyberGreen and ROX as reference dyes (Invitrogen). Samples were normalized using GAPDH primers. Both target gene and GAPDH amplification reactions approached 100% efficiency as determined by standard curves. Three independent biological samples were analyzed, and one representative set of results is shown. Primer sequences were as follows: USP1 (Forward): 5’- GGACGCGTTGCTTGGAATGT-3’ (Reverse) 5’-TGCCCATCTCAGGGTCTTCA-3’. GADPH: (Forward) 5’-AGCCTCCCGCTTCGCTCTCT-3’ (Reverse) 5’-GAGCGATGTGGCTCGGCTGG-3’ .Transfected cells were replated at low density. 24 hr after replating cytochalasine B (4.5 μg/ml-Sigma) was added to the media and 40 hr later cells were washed 1 min with hypotonic buffer (KCl 0.0075 M) diluted 1/10 from stock solution in PBS 1X, twice with PBS 1X and fixed with PFA/sucrose 2% for 20 min. DAPI staining served to visualize cell nuclei. 300 binucleated cells were analyzed.Cells were treated with 0.1 μg/ml colcemid (Gibco) for 3 hr. Cells were trypsinized for 1 min trying to take off only the mitotic cells. After centrifugation cell pellets were resuspended in hypotonic solution (0.075 M KCl) and incubated for 15 min at 37°C. Samples were then fixed in a methanol/acetic acid solution (3:1), washed three times in the fixative solution and dropped on slides to obtain spread chromosomes. Metaphase slides were incubated with Rnase (10 μg/ml in 2× SSC) for 1 hr at 37°C followed by dehydration in successive ethanol baths . The RP 11 36B6 BAC (corresponding to FRA7H locus) was labeled by nick translation (Abbott) with green dUTP (Vysis Spectrum) and ethanol precipitated with human Cot-1 DNA  overnight at −20°C. Precipitated DNA was incubated for 1 hr at 37°C in hybridization mix. The probe was denatured for 8 min at 70°C and applied to denatured metaphases. Samples were incubated over night at 37°C and, after washing  and mounted in VECTASHIELD Antifade Mounting Medium with DAPI. The images were acquired by using an inverted wide-field Zeiss Axio Observer Z1 microscope fitted with a x63 oil NA 1.4 objective and an Axiocam MRm CDD camera.Frequency distributions of DNA track length were determined with GraphPad Prism 5 software. In non-Gaussian distributions, Mann–Whitney and Kruskal–Wallis tests were used for statistical analyses when comparing two and more than three variables, respectively. Other statistical analyses were performed with GraphPad in Stat software using the Student’s t test and the one-way ANOVA test when applicable. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Tony Hunter as the Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Cyclin kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers all found the hypothesis that p21 has a basal role in unstressed cells at the replication fork to keep the Alt polymerases from functioning by direct interactions to be important, and the data showing that such a process is working in cells established for growth in cell culture to be compelling. However, for publication it would be essential to show:1) That this role is also key in primary cells, and2) To test the hypothesis further it is key to show that a PIP domain mutation in p21 (ablating interaction sites) cannot complement a null mutation of the endogenous p21 alleles. This experiment could be done in either human or mouse cells.The reviewers’ comments below are given to provide you with justifications for these issues and have other points that you may wish to address for improving the manuscript in a revision.Reviewer #1:eLife but feel that there are still some additional experiments that need to be done that will make the story more compelling. The key new concept here is that p21 has a basal function in non-stressed cells by interfering with a trans-lesion DNA polymerase κ. In the main system used, an established cell line, the data for a basal activity seems compelling. However, it is essential to show that similar phenotypes for the PIP mutant of PCNA are detected in primary cells or in any convincing way that would eliminate the suggestion that this role for p21 in fact arises because of the initial stress and adaption to cell culture. Technically the authors should also provide more data to show that their siRNA knock-downs are specific or use better still use a knock out PCNA strain (by CRISPR/CAS) with a replacement of the gene with a mutant p21.I found the paper interesting and certainly of potential high impact. So I would like to recommend publication in Following up on the possibility of selection or adaption for the phenotype, it might be worthwhile for the authors to suggest or discuss how having an alternative polymerase working excessively at forks (without p21) might lead to more ori firing and shorter replication tracks in the first place. Clearly, and just one proposal would be that the "alt polymerase" leads to mistakes and damage stressing the daughter cells with replication pausing which in turn evolves to the need for more ori firing. But is that consistent with what we know about the effects of Pol Κ at the replisome? Might other polymerases be more effective in producing double strand breaks and stress? Or do the authors have a way to explain how it comes to be that more ori's are activated.Reviewer #2:Mansilla et al. describe a novel function of p21 in preventing genomic instability associated with DNA replication. The authors show that the WT p21, but not the PIP mutant p21 colocalizes with GFP-PCNA. They show that p21 depletion in U2OS and HCT116 cells results in shorter replication tracks without application of exogenous DNA damage and increased origin firing that presumably compensates for the shorter tracks. Cells with decreased levels of p21 also have increased genome instability. Many of the phenotypes were reversible by complementation with WT but not the PIP mutant of p21 or by concomitant depletion of Pol κ. This led the authors to propose that the low levels of p21 present in cells without any exogenous DNA damage and p53 induction are there to protect the PCNA from engaging Pol κ which would result in less processive replication and induction of genomic instability.This is potentially an interesting set of observations. However, the study is largely done with one cell line, using one siRNA against p21 and Pol κ. Although complementation was performed with p21 cDNA, the key observations should be repeated in p21-/- MEFs or human primary p21-null cells. The most interesting aspect of the study is the rescue of the phenotypes of p21 depletion by Pol κ depletion, but in order for these to be convincing, additional data would be needed. The Pol κ depletion experiments were not done in HCT116 cells and even the efficiency of depletion of Pol Κ was shown only on the exogenously expressed GFP-tagged Pol κ. At least RT-PCR data for Pol κ should be included. In addition, it would be important to show that the levels of Pol κ are increased at the fork in cells lacking p21. This can be done using iPOND with and without p21 depletion.Reviewer #3:This manuscript presents evidence that the interaction of p21 with PCNA recruits p21 to replication factories, that in the absence of external DNA damage, deletion of p21 impairs normal replication and increases replication stress, that p21 prevents Pol κ  from contributing to the replication of undamaged DNA, and that these effects depend on the PCNA interacting region domain of p21, but not its CDK-binding domain. These observations are consistent with the authors' hypothesis that p21 has a role in normal replication via an interaction with PCNA. This implies a novel role for p21. In my opinion, the manuscript is well written and should stimulate new studies, and I believe it is worth publishing.That said, the results obtained here in U2OS and HCT116 cells lines have not yet been confirmed using primary cells, and the evidence for the role of pol κ as the major DNA polymerase involved does not yet exclude roles for several other "alt" polymerases not examined here. These two limitations should be acknowledged, and the language used to express the authors' main claims could be modified at several places ; Discussion, "Pol κ is the most processive" – not so sure, e.g., what about ζ?) […] The reviewers all found the hypothesis that p21 has a basal role in unstressed cells at the replication fork to keep the Alt polymerases from functioning by direct interactions to be important, and the data showing that such a process is working in cells established for growth in cell culture to be compelling. However, for publication it would be essential to show:1) That this role is also key in primary cells, and2) To test the hypothesis further it is key to show that a PIP domain mutation in p21 (ablating interaction sites) cannot complement a null mutation of the endogenous p21 alleles. This experiment could be done in either human or mouse cells.Primary human cells were used as suggested by the three reviewers and the editor. We used primary human foreskin fibroblast and mesenchymal cells from umbilical cord. As in tumor cells, in such cellular models p21 promotes nascent DNA elongation preventing the accumulation of replication stress and genomic instability markers. This is shown in current We also provide data demonstrating that only the wt but not the PIP domain mutant complements a null mutation of the endogenous p21 allele. This is showed in The reviewers’ comments below are given to provide you with justifications for these issues and have other points that you may wish to address for improving the manuscript in a revision.We understand the concerns raised by reviewers and therefore we have addressed each one of their comments. In fact, in order to address reviewers’ comments we have generated 2 Figures  and modiReviewer #1:I found the paper interesting and certainly of potential high impact. So I would like to recommend publication in eLife but feel that there are still some additional experiments that need to be done that will make the story more compelling. The key new concept here is that p21 has a basal function in non-stressed cells by interfering with a trans-lesion DNA polymerase κ. In the main system used, an established cell line, the data for a basal activity seems compelling. However, it is essential to show that similar phenotypes for the PIP mutant of PCNA are detected in primary cells or in any convincing way that would eliminate the suggestion that this role for p21 in fact arises because of the initial stress and adaption to cell culture.As specified above, we have performed the experiments in two primary cells and the data is shown in current Technically the authors should also provide more data to show that their siRNA knock-downs are specific or use better still use a knock out PCNA strain (by CRISPR/CAS) with a replacement of the gene with a mutant p21.We understand reviewer´s 1 concern. It is true that we have used only one siRNA for p21. However, we also obtained similar results in HCT116 cells in which p21 has been eliminated by homologous recombination . MoreoveWe however believe that reviewer´s 1 concern should be addressed also for the pol κ siRNA. In this case we used a second siRNA for pol κ and observed similar results to those obtained with the first siRNA specific for pol κ . We alsoFollowing up on the possibility of selection or adaption for the phenotype, it might be worthwhile for the authors to suggest or discuss how having an alternative polymerase working excessively at forks (without p21) might lead to more ori firing and shorter replication tracks in the first place. Clearly, and just one proposal would be that the "alt polymerase" leads to mistakes and damage stressing the daughter cells with replication pausing which in turn evolves to the need for more ori firing. But is that consistent with what we know about the effects of Pol Κ at the replisome? Might other polymerases be more effective in producing double strand breaks and stress? Or do the authors have a way to explain how it comes to be that more ori's are activated.While the tight association between increased origin firing and reduction in fiber length is well documented in many reports, it is yet unclear which of these two events precede and triggers the other replication defect . For example, in the case of Chk1 depletion, the mechanism may involve an initial dysregulation of origin firing which causes a depletion of dNTPs available for nascent DNA elongation . In contrast, we postulate that the trigger for replication defects in cells depleted from p21 is not origin firing. We propose so because pol κ depletion fully complements the replication defects caused by p21 depletion. Given that the function of pol κ is tightly associated with the elongation of nascent DNA (and not with origin firing), we believe that in the case of this report it is more likely that the primary defect in p21 depleted samples is the slow elongation of nascent DNA at replication forks which then triggers increased origin firing as an attempt to finalize DNA replication of DNA regions which are being duplicated with a slow rate. In fact, we also speculate that such increased origin firing may prevent genomic instability to a certain extent. While the number of slow elongating fibers is frequent (they would not be detected by the stretching assay otherwise), the accumulation of genomic instability events is much less frequent  supporting a global change in DNA replication speed rather that the random collision of replication forks with DNA damage. Second, we did not detect phospho-Chk1 accumulation (not shown in the manuscript),  which inReviewer #2:Mansilla et al. describe a novel function of p21 in preventing genomic instability associated with DNA replication. The authors show that the WT p21, but not the PIP mutant p21 colocalizes with GFP-PCNA. They show that p21 depletion in U2OS and HCT116 cells results in shorter replication tracks without application of exogenous DNA damage and increased origin firing that presumably compensates for the shorter tracks. Cells with decreased levels of p21 also have increased genome instability. Many of the phenotypes were reversible by complementation with WT but not the PIP mutant of p21 or by concomitant depletion of Pol κ. This led the authors to propose that the low levels of p21 present in cells without any exogenous DNA damage and p53 induction are there to protect the PCNA from engaging Pol κ which would result in less processive replication and induction of genomic instability.This is potentially an interesting set of observations. However, the study is largely done with one cell line, using one siRNA against p21 and Pol κ. Although complementation was performed with p21 cDNA, the key observations should be repeated in p21-/- MEFs or human primary p21-null cells.Reviewer 2 is right and his/her request is in agreement with the rest of the reviewers and the editor. As discussed above these experiments were performed in two primary human cells and are summarized in The most interesting aspect of the study is the rescue of the phenotypes of p21 depletion by Pol κ depletion, but in order for these to be convincing, additional data would be needed. The Pol κ depletion experiments were not done in HCT116 cells and even the efficiency of depletion of Pol Κ was shown only on the exogenously expressed GFP-tagged Pol κ. At least RT-PCR data for Pol κ should be included.In the current version of our manuscript we have included an RT-PCR analysis see . Also, wIn addition, it would be important to show that the levels of Pol κ are increased at the fork in cells lacking p21. This can be done using iPOND with and without p21 depletion.We believe that this point of reviewer 2 is important. Hence we used two experimental approaches to answer this question. First, we evaluated the interaction of PCNA and GFP-pol κ in the chromatin fraction and found increased interaction of pol κ and PCNA in the absence of p21 . Second,Reviewer #3:This manuscript presents evidence that the interaction of p21 with PCNA recruits p21 to replication factories, that in the absence of external DNA damage, deletion of p21 impairs normal replication and increases replication stress, that p21 prevents Pol κ  from contributing to the replication of undamaged DNA, and that these effects depend on the PCNA interacting region domain of p21, but not its CDK-binding domain. These observations are consistent with the authors' hypothesis that p21 has a role in normal replication via an interaction with PCNA. This implies a novel role for p21. In my opinion, the manuscript is well written and should stimulate new studies, and I believe it is worth publishing.That said, the results obtained here in U2OS and HCT116 cells lines have not yet been confirmed using primary cells.As discussed before this particular point has been raised by the three reviewers and has been addressed using two primary cell lines: human foreskin fibroblast and mesenchymal cells from the umbilical cord see .And the evidence for the role of pol κ as the major DNA polymerase involved does not yet exclude roles for several other "alt" polymerases not examined here. These two limitations should be acknowledged.Our point was that the replication stress and genomic instability outputs evaluated in this manuscript were all reverted (surprisingly) by the depletion of one single DNA polymerase, pol κ. We believe that it is possible that other replication associated defects caused by p21 depletion (not explored in this study) could depend on another alternative polymerase. In fact, when using methanol/acetone extraction to reveal alterative pols associated with replication factories , we detected increased focal organization of pol κ, η, ι but not in Rev1 (see below). The difference between Rev1 and the other pols may depend on the lack of PIP box in Rev1 . Notably, pol κ is the alt pol with the lowest recruitment to replication factories in control cells (grey bars), and the one with highest fold induction after p21 depletion . In any And the language used to express the authors' main claims could be modified at several places ; Discussion, "Pol κ is the most processive" – not so sure, e.g., what about ζ?)We have modified the above mentioned sentences according to reviewer´s 3 request (see changes in the subsection “p21 prevents the aberrant use of the alternative DNA polymerase κ during the replication of undamaged DNA”, last paragraph; subsection “The PCNA-binding domain of p21 is necessary and sufficient to prevent the replication defects introduced by Pol κ” and subsection “The complex regulation of Pol κ by p21”.