Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers

Špela Zemljič Jokhadar; Biljana Stojković; Marko Vidak; Tjaša Sorčan; Mirjana Liovic; Marcos Gouveia; Rui D. M. Travasso; Jure Derganc

doi:10.1371/journal.pone.0231606

Peer Review History

Original SubmissionMarch 25, 2020
1 Jun 2020 Decision Letter - Jung Weon Lee, Editor PONE-D-20-08552 Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers PLOS ONE Dear Dr. Zemljič Jokhadar, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Two experts have reviewed the manuscript and found that the current study needs to be more clarified with more controls and qualified images enough to be published in the journal. Please submit your revised manuscript by Jul 16 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Jung Weon Lee, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please provide additional information about the NEB1, KEB7, NHEK2 and MDA-MB-231 cell lines used in this work, including the source of the cell lines and any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The cytokeratin pattern of human skin keratinocytes is well known. Despite a series of publications the exact influence of cytokeratins on cellular mechanical properties needs further elucidation to understand their functional role. The authors isolated primary cells from healthy persons (NHEK2 ), immortalized wild type cells (NEB1) and isolated and immortalized cells (KEB7) with K14 R125P mutant. Using optical tweezers a small glass ball is moved towards one side of these cells at two different speeds. The shift of the ball out of the center of the beam gives a measure of the force which is applied to the cell by the touching ball. This procedure is performed once or twice at the same position. The stiffness as determined by this indentation differs between the three cell types, being highest in the primary cells and lowest in the immortalized cells, slightly higher in the mutant cells. The values of the immortalised cells vary significantly less than those of the other cells, including fixed cells which act as controls. The slow moving bead is facing higher stiffness than when approached fast (0,1 versus 1 µm/s) The measurements are not sensitive to cytochalasin. The biomechanical measurements are convincing and elegant. Whether the cells are observed using bright field microscopy and not phase contrast or DIC remains an open question. Better optical contrast would have allowed to follow cell behaviour clearly and the size of the bead will be hidden by refraction anyway. The main shortcomings of this manuscript is the lack of functional aspects, on the role of the various cytoskeletal elements for these mechanical properties. At least some good fluorescence images of F-actin, cytokeratin and microtubules would be required to see what is going on on the sides where the ball is touching. This area strongly depends on adhesivity of the cells and the speed of cell movement. The cortical actin layer may be almost missing and F-actin may appear in form of ordered bundles (fast movement and strong adhesion at rear end), or in very slowly moving cells a well developed cortical net of fine fibrillar actin may be apparent. How close are the cytokeratins in these areas, are the different cell types moving at different speed and do they keep direction? In case the cells would be turning around the cortical stiffness strongly depends on the direction of the turn as was shown by acoustic microscopy of keratinocytes. Reviewer #2: Most of eukaryotic cells lack a cell wall and the main determinant of the cell surface stiffness is represented by cellular cortex which is a thin crosslinked actin network that lies immediatly under the plasma membrane. In the last years the cortex has received increasing attention due to its functions in many cellular processes including cytokinesis and cell migration. Local mechanical probing has shown that actin rules cortical stifness whereas intermediate filaments, namely vimentin, contribute mainly to cytoplasmic stifness. In the manuscript by Zemljic s. et al. the authors attempted to assess the role played by keratin filaments in regulating the keratinocytes cortical stifness by employing a unusual approach involving the use of lateral indentation with optical tweezers. They compared the cortical stifness of three different keratinocyte cell lines alongside to their aptitude to tune focal adhesions by quantifying the bead adhesion events in indentation experiments. Prior publications there are some major and minor demanding issues that have to be sorted-out. Major 1) The three cell lines displayed different cortical stiffness. However, as the authors mentioned, the cell lines are quite different being 2 out of three immortalized and one primary keratinocytes (NHEK2). Could you please provide a Western Blot showing the amount of Keratin 14 for each cell line when compared to a suitable control? 2) The authors state that keratins have been shown to partecipate in focal adhesion thus they assess ahether the mutated keratin affect the process they quantified the adhesion events in indentation experiments by using beads coated with anti-integrin B1 antibody. Would it be possible to coat the beads with an extracellular matrix component (e.g. collagen) which represents the physiological ligand for integrin? 3) Table S1 and fig.3: while for NEB1 cells measurements are quite clusterd for the others they are quite spread. Could you please implement data for NHEK2 cells at the condition of 1um/s and those of fixed NEB1with at least 35 measurements? Furthermore, would it be possible implement the experiment by increasing twice the indentation deep (400 nm instead of 200 nm)? 4) I understand that the crosstalk between inetermediate- and actin-filaments is mostly unknown. However, it would be greatly appreciated whether the authors could discuss a little bit this aspect in the discussion. Minor 1)Concerning the material and methods section the author should provide details regarding the medium used to culture cells. a) Does the EpiLife medium contain, or not, calcium? if yes how much? b) How much is diluted the EpiLife defined growth supplement? Is plasticware coated? if yes what with has been coated? 2)Few typos. 3)When it comes to the section "Results" (pag. 7 line 172) could you please clarify what is the meaning of k1 and k2. 4)It may be that I missed that point but for how long and what is the concentration that has been used for CytochalisnD experiment? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Juergen Bereiter-Hahn, Institute for Neurosciences and Cell Biology, Goethe University Frankfurt Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0231606.r001
Revision 1
18 Sep 2020 Author Response Reviewer #1: The cytokeratin pattern of human skin keratinocytes is well known. Despite a series of publications the exact influence of cytokeratins on cellular mechanical properties needs further elucidation to understand their functional role. The authors isolated primary cells from healthy persons (NHEK2 ), immortalized wild type cells (NEB1) and isolated and immortalized cells (KEB7) with K14 R125P mutant. Using optical tweezers a small glass ball is moved towards one side of these cells at two different speeds. The shift of the ball out of the center of the beam gives a measure of the force which is applied to the cell by the touching ball. This procedure is performed once or twice at the same position. The stiffness as determined by this indentation differs between the three cell types, being highest in the primary cells and lowest in the immortalized cells, slightly higher in the mutant cells. The values of the immortalised cells vary significantly less than those of the other cells, including fixed cells which act as controls. The slow moving bead is facing higher stiffness than when approached fast (0,1 versus 1 µm/s) The measurements are not sensitive to cytochalasin. 1. The biomechanical measurements are convincing and elegant. Whether the cells are observed using bright field microscopy and not phase contrast or DIC remains an open question. Better optical contrast would have allowed to follow cell behaviour clearly and the size of the bead will be hidden by refraction anyway. We thank the reviewer for the positive comments. Our current optical tweezers set-up only supports normal bright field imaging. Fortunately, even with this technique the contrast was good enough so that most of the time we were able to perform the indentation experiments by avoiding lamellipodia that would interfere with the measurement. For our future studies, we would like to upgrade the microscope with an external phase contrast module, which would be compatible with the laser and would thus expand the usability of the set-up. The missing information about the employed microscopy technique has been now added to Materials and methods and in the Figure caption. 2. The main shortcomings of this manuscript is the lack of functional aspects, on the role of the various cytoskeletal elements for these mechanical properties. At least some good fluorescence images of F-actin, cytokeratin and microtubules would be required to see what is going on on the sides where the ball is touching. This area strongly depends on adhesivity of the cells and the speed of cell movement. The cortical actin layer may be almost missing and F-actin may appear in form of ordered bundles (fast movement and strong adhesion at rear end), or in very slowly moving cells a well developed cortical net of fine fibrillar actin may be apparent. How close are the cytokeratins in these areas, are the different cell types moving at different speed and do they keep direction? In case the cells would be turning around the cortical stiffness strongly depends on the direction of the turn as was shown by acoustic microscopy of keratinocytes. We thank the reviewer for the observation that the information on the state of all cytoskeletal elements is crucial for interpretation of mechanical properties of cells. While a detailed scrutiny of the interplay between different cytoskeletal elements is beyond the scope of this paper (and, in fact, our experimental capacity), we performed confocal imaging to visually examine the integrity of the cytoskeletal elements in our cells. These new results are now presented in a new section »Distribution of cytoskeletal elements« with additional figures (Fig 2 and Fig S2), and a description of immunofluorescence is added to Materials and methods. The results showed that all the cell lines exhibited the expected behavior, i.e., the keratin filaments were disrupted only in the mutant keratin cell line KEB7. Also, within the resolution of our confocal microscope, actin filaments and microtubules exhibited normal morphology and the sub-membrane actin was present at the point of the bead indentation in all three cell lines. We thank the reviewer for pointing out the relation between cell migration and their mechanical properties, which we did not explicitly consider in our first submission. As noted by the reviewer, the leading edge of migrating keratinocytes markedly differs from their trailing edge in terms of cell stiffness (Bereiter-Hahn & Lüers, 1998) and membrane tension (Lieber et al., 2015). In our indentation experiments, which were performed within approximately 30 min, we did not observe notable cell migration with any cell line. In fact, very few cells exhibited the typical morphology of migrating cells, i.e., the one resembling a fried egg cut into a crescent shape with distinct leading/trailing edges. To examine cell behavior over longer periods of time we have now also performed time-lapse recording of cells. These experiments confirmed that, unlike the frog and fish keratinocytes that were used in the above studies, our cells did not exhibit notable movements and rather extended lamellipodia in all directions. These points are now emphasized in Discussion together with additional references. To avoid misunderstanding, we changed the terms leading/trailing edge to a more descriptive ones (e.g., “trailing edge” -> “cell side without lamellipodia”). Time-lapse recordings of typical cells have been added to the Supporting information. Also, we now clearly state that out mechanical experiments were performed on individual non-confluent cells. Following the suggestions made by the reviewer, we further investigated a possible connection between the mutated keratin and the cortical stiffness. Namely, previous studies on live GFP-transfected mutant keratinocytes found highly dynamic keratin particles in the cell periphery, that could in principle interact with the cortex [refs 7-10]. We therefore performed additional confocal microscopy to compare distributions of actin and keratin in NEB1 and KEB7 cells. The results are presented in the Supporting material (Fig S3). In both cell types, the keratin signal was distributed in the cell interior underneath the actin cortex. Within our optical resolution, no larger keratin particles and no clear interactions between the keratin and the cortex were detected. These aspects are now further addressed in the Discussion (see also the answer to the comment #4 made by reviewer #2). Additional references that were added, regarding the mechanics of migrating keratinocytes: Bereiter-Hahn J, Lüers H. Subcellular tension fields and mechanical resistance of the lamella front related to the direction of locomotion. Cell Biochem Biophys. 1998;29(3):243-62. Epub 1998/12/30. doi: 10.1007/bf02737897. PubMed PMID: 9868581. Lieber AD, Schweitzer Y, Kozlov MM, Keren K. Front-to-rear membrane tension gradient in rapidly moving cells. Biophys J. 2015;108(7):1599-603. Epub 2015/04/12. doi: 10.1016/j.bpj.2015.02.007. PubMed PMID: 25863051; PubMed Central PMCID: PMCPMC4390806. Reviewer #2: Most of eukaryotic cells lack a cell wall and the main determinant of the cell surface stiffness is represented by cellular cortex which is a thin crosslinked actin network that lies immediatly under the plasma membrane. In the last years the cortex has received increasing attention due to its functions in many cellular processes including cytokinesis and cell migration. Local mechanical probing has shown that actin rules cortical stifness whereas intermediate filaments, namely vimentin, contribute mainly to cytoplasmic stifness. In the manuscript by Zemljic s. et al. the authors attempted to assess the role played by keratin filaments in regulating the keratinocytes cortical stifness by employing a unusual approach involving the use of lateral indentation with optical tweezers. They compared the cortical stifness of three different keratinocyte cell lines alongside to their aptitude to tune focal adhesions by quantifying the bead adhesion events in indentation experiments. Prior publications there are some major and minor demanding issues that have to be sorted-out. Major 1. The three cell lines displayed different cortical stiffness. However, as the authors mentioned, the cell lines are quite different being 2 out of three immortalized and one primary keratinocytes (NHEK2). Could you please provide a Western Blot showing the amount of Keratin 14 for each cell line when compared to a suitable control? We thank the reviewer for the constructive remarks regarding our study and we acknowledge that western blot of keratins is important information that is needed to interpret the results of mechanical experiments. We therefore performed western blot showing keratin K14 in all three utilized cell lines (in our study, NEB1 cells served as control cells as the main focus was to measure if the impaired keratin affects the cortical stiffness). The results showed that NEB1 and NHEK2 cells had much more keratin 14 than the mutant KEB7 cell (Fig S4). We quantified these results as NHEK2 >NEB1>KEB7, where there is about 30% less keratin in NEB1 cells than in NHEK2, and about 10x less keratin in KEB7 cells than in NEB1. The western blot has been now added to the Supporting information (Fig S4). These new results support the implicit assumption that was made in our first submission, i.e., that the major difference between cell lines is the severely impaired keratin in mutant cells. These new data is in line with the main result of our study, namely that keratin is not a major player in cortical stiffness. 2. The authors state that keratins have been shown to partecipate in focal adhesion thus they assess ahether the mutated keratin affect the process they quantified the adhesion events in indentation experiments by using beads coated with anti-integrin B1 antibody. Would it be possible to coat the beads with an extracellular matrix component (e.g. collagen) which represents the physiological ligand for integrin? We thank the reviewer for this recommendation. In our study we anticipated that the side-indentation of cells with optical tweezers could be useful for assessing cell-bead adhesion. We therefore employed the anti-integrin B1 antibody because we could covalently attached it to our streptavidin-coated silica beads via a biotin-streptavidin bond. Following the reviewer’s suggestion we explored also the possibility to coat our beads with collagen or fibronectin via non-specific binding, but in the given time-frame we could not develop a robust experimental protocol that would ensure reliable experimental results (e.g., we were not able to develop a reliable control experiment for assessing the extent of coating). Encouraged by the reviewer’s comment, we are now even more convinced that our protocol would be particular suitable for adhesion experiments because the side-indentation into a cell is gentle and can be well controlled. We are therefore developing an upgraded tweezers set-up with real-time force measurement and feed-back that would allow a better control over cell-bead contact times (see also our response to the next question) and look forward to implementing also alternative bead coatings. 3. Table S1 and fig.3: while for NEB1 cells measurements are quite clusterd for the others they are quite spread. Could you please implement data for NHEK2 cells at the condition of 1um/s and those of fixed NEB1 with at least 35 measurements? Furthermore, would it be possible implement the experiment by increasing twice the indentation deep (400 nm instead of 200 nm)? We acknowledge that there is a seemingly puzzling discrepancy in the number of the measured cells for each cell type. The reason for it lies in our experimental protocol, which proved to be quite elaborate and time consuming. Namely, in the presented experimental set-up, the force on the bead was not measured in real time but only after the indentation was completed. We therefore could not implement a feed-back mechanism that would automatically retract the bead when the force (or indentation depth) reached a certain value. The contact point between the bead and the cell was adjusted manually, based on real-time visual inspection (in the experiment, the bead was moving to-and-fro a predefined point with a constant speed and the cell was slowly moved towards the same predefined point using a manual the microscope stage until the bead started touching the cell). Because we aimed to indent the cells as gently as possible (to be able to repeat two measurements on the same spot), many of the trials ended without a usable force-indentation curve. In addition, many of the indentations had to be discarded because free-floating debris, which is always present with living cells, was attracted into the laser trap and ruined the force measurement. To assure the same experimental conditions for all three cell lines, the experiments were carried in parallel in the same time period. We aimed to collect approximately 20 data points for each cell, which is comparable to the number of data points that are typically collected in similar studies. However, it turned out that the primary cell line was much more difficult to work with, and by the time we had collected 20 points for NHEK2 cells, we ended up with many more data points on the NEB1 and KEB7 cell lines. Fortunately, this, in turn, vastly improved the statistics for these two cell types, which were in the focus of our study. We have now amended the manuscript to emphasize the above-mentioned drawbacks of our experimental protocol. Also, we are now developing an enhanced protocol with a real-time force measurement that would greatly improve our experimental throughput and provide a much better control over the maximum indentation depth (or maximum indentation force). Such a protocol would also allow to reliably acquire and analyze the force-indentation curves at greater indentation depths, as suggested by the reviewer. 4. I understand that the crosstalk between inetermediate- and actin-filaments is mostly unknown. However, it would be greatly appreciated whether the authors could discuss a little bit this aspect in the discussion. We agree with both reviewers (see also the comment #2 by reviewer #1) that the ultimate goal of studies like ours should be a better understanding of the functional role of keratins in cell mechanics. As described in our answer to the comment #2 by reviewer #1, we performed additional imaging to explore possible connections between actin and keratin. These aspects are now further addressed in the Discussion. A reference to a recent review paper on the cytoskeletal cross-talk has been added (Seetharaman et al. 2020). Seetharaman, Shailaja, and Sandrine Etienne-Manneville. "Cytoskeletal crosstalk in cell migration." Trends in Cell Biology (2020). Minor 1. Concerning the material and methods section the author should provide details regarding the medium used to culture cells. a) Does the EpiLife medium contain, or not, calcium? if yes how much? b) How much is diluted the EpiLife defined growth supplement? Is plasticware coated? if yes what with has been coated? The missing data was added to the manuscript. 2. Few typos. The manuscript was proofread by a professional English speaker. 3. When it comes to the section "Results" (pag. 7 line 172) could you please clarify what is the meaning of k1 and k2. The missing data was added to the manuscript. 4. It may be that I missed that point but for how long and what is the concentration that has been used for CytochalisnD experiment? The missing data was added to the manuscript. https://doi.org/10.1371/journal.pone.0231606.r002
6 Oct 2020 Decision Letter - Jung Weon Lee, Editor PONE-D-20-08552R1 Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers PLOS ONE Dear Dr. Zemljič Jokhadar, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. One of the previous reviewers found that certain minor points should clarified before a publication in the journal including typos. Please submit your revised manuscript by Nov 20 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Jung Weon Lee, Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: (No Response) Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Fig 2 shows a KEB7 cell with very sparse F-actin, this does not support the statement line 196/197 that the cytoskeleton of these cells differs only in the lack of keratin 14 expression. However in S3 a KEB7 cell is shown that expresses well developed F-actin. I propose to mention this variability in the legend to Fig. 2. The final question of the role of cytokeratin in cell mechanics has not been solved and will require exensive further work. The very high variability of the reaction data would support the hypothesis that cytokeratin stabilises F-actin arrangements and by this supports more homogenous reactions. But anyway, this is a very crude hypothesis which also needs intensive research. Therefore we can accept that the paper does not try to extend our knowledge by extensive hypotheses. Reviewer #2: I truly appreciated the efforts made by Jokhadar and colleagues and I understand that they encountered some kind of technical difficulties in setting up robust protocol, due to the time window, to properly address the arguments raised by the reviewer. On the whole the manuscript has been improved. However, in my opinion, before publication a couple of minor issues should be settled. 1) When comparing protein abundance across different samples (e.g. different cell lines), it is critical to have a method to account for variation due to errors in loading or later during the transfer. Thus, I am wondering how can be possible that the authors could estimate the amount of a given protein in a Western Blot without of a proper loading control? Please provide a proper loading control (e.g. GAPDH, actin, tubulin, lamin, vinculin etc…). Only after having assessed the band intensities of both proteins (housekeeping and keratin 14) one can draw conclusions and compare the protein amount among different samples. 2) Please check once more the manuscript because throughout the main text there are still few typos that should be edited (e.g. 12.5 rpm -> either 12.5 Krpm or 12,500 rpm) 3) In the section material and methods it would be nice to indicate the catalogue number in addition to the company from which the authors purchased the antibodies used in the study. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Juergen Bereiter-Hahn Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0231606.r003
Revision 2
10 Nov 2020 Author Response Reviewer #1 Fig 2 shows a KEB7 cell with very sparse F-actin, this does not support the statement line 196/197 that the cytoskeleton of these cells differs only in the lack of keratin 14 expression. However in S3 a KEB7 cell is shown that expresses well developed F-actin. I propose to mention this variability in the legend to Fig. 2. The final question of the role of cytokeratin in cell mechanics has not been solved and will require exensive further work. The very high variability of the reaction data would support the hypothesis that cytokeratin stabilises F-actin arrangements and by this supports more homogenous reactions. But anyway, this is a very crude hypothesis which also needs intensive research. Therefore we can accept that the paper does not try to extend our knowledge by extensive hypotheses. We thank the reviewer for pointing out this discrepancy. After reviewing the data, we realized that the weak intensity of actin signal in KEB7 cells in Fig. 2 was a result of an error in transferring the raw images into a single panel. We therefore re-edited Fig. 2 and double-checked that all the images appear with their original intensity. Nevertheless, we mentioned in the results (line 200) that the distribution of the cytoskeletal elements differs moderately even among cells of the same cell type. We also added to the legend of Fig 2 that the images were taken at the height were the bead indents the cell, which is approximately 5 µm above the bottom of the chamber. Reviewer #2 1) When comparing protein abundance across different samples (e.g. different cell lines), it is critical to have a method to account for variation due to errors in loading or later during the transfer. Thus, I am wondering how can be possible that the authors could estimate the amount of a given protein in a Western Blot without of a proper loading control? Please provide a proper loading control (e.g. GAPDH, actin, tubulin, lamin, vinculin etc…). Only after having assessed the band intensities of both proteins (housekeeping and keratin 14) one can draw conclusions and compare the protein amount among different samples. We thank the reviewer for this constructive remark and we repeated the WB once more with GAPDH as the control. As we had three independent experiments now, the results present the mean over all of them. 2) Please check once more the manuscript because throughout the main text there are still few typos that should be edited (e.g. 12.5 rpm -> either 12.5 Krpm or 12,500 rpm) We checked the manuscript for the typos (the mentioned numbers were corrected to 12,500 rpm). 3) In the section material and methods it would be nice to indicate the catalogue number in addition to the company from which the authors purchased the antibodies used in the study. We added the catalogue numbers of the antibodies used. https://doi.org/10.1371/journal.pone.0231606.r004
25 Nov 2020 Decision Letter - Jung Weon Lee, Editor PONE-D-20-08552R2 Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers PLOS ONE Dear Dr. Zemljič Jokhadar, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. One of the previous reviewers still commented that certain points in the revised manuscript should be clarified for a publication in the journal. Please submit your revised manuscript by Jan 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: I acknowledge the efforts made by the authors. Indeed, the authors fulfilled the reviewer’s requirements. However, I still noticed that there are a couple of minor concerns, looking like kind of discrepancies, that are deserving some attention. Examples are given below: 1) Lines 218-224: the figures provided by the authors about Western Blot keratin 14 quantification differ from those that appear in the Supporting Information legend to figure S4 (lines 544-547). Which one should I trust? 2) In the section material & methods when it comes the “Protein extraction and Western blotting” line 117 it is not clear whether the SDS-PAGE is a gradient gel (4-12%) or it is a 20% SDS-PAGE. Please clarify these minor issues. ****** 7. 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Revision 3
1 Dec 2020 Author Response We would like to thank the Reviewer #2 for his very thorough overview of our manuscript. Both mistakes he pointed out were the results of our inaccuracy. • Lines 218-224: the values written in these lines are correct and represent the mean of three independent Western Blots we made. In the lines 544-547 we forgot to update the numbers, which we corrected now. • Line 117: SDS-PAGE is a gradient gel (4-12%) and not a 20% SDS-PAGE, which we corrected now. Attachments Attachment Submitted filename: Answers to the Reviewer_.docx https://doi.org/10.1371/journal.pone.0231606.r006
7 Dec 2020 Decision Letter - Jung Weon Lee, Editor Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers PONE-D-20-08552R3 Dear Dr. Zemljič Jokhadar, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jung Weon Lee, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: I am very grateful to the authors for their endless efforts aimed to improve their manuscript. The minor issues that deserved attention have now been properly addressed. Hence, their manuscript is now worthy of publication in the PLOS ONE Journal. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No https://doi.org/10.1371/journal.pone.0231606.r007
Formally Accepted
21 Dec 2020 Acceptance Letter - Jung Weon Lee, Editor PONE-D-20-08552R3 Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers Dear Dr. Derganc: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Jung Weon Lee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0231606.r008

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