Piezo1 balances membrane tension and cortical contractility to stabilize intercellular junctions and maintain epithelial barrier integrity

  1. Ahsan Javed
  2. Aki Stubb
  3. Clémentine Villeneuve
  4. Franziska Peters
  5. Matthias Rübsam
  6. Carien M. Niessen
  7. Leah C. Biggs
  8. Sara A. Wickström

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Formation of a bi-directional skin barrier is essential for organismal survival and maintenance of tissue homeostasis. Barrier formation requires positioning of functional tight junctions (TJ) to the most suprabasal viable layer of the epidermis through a mechanical circuit that is driven by generation of high tension at adherens junctions. However, what allows the sensing of tension build-up at these adhesions and how this tension is balanced to match the requirements of tissue mechanical properties is unclear. Here we show that the mechanosensitive ion channel Piezo1 is essential for the maturation of intercellular junctions into functional, continuous adhesions. Deletion of Piezo1 results in an imbalance of cell contractility and membrane tension, leading to a delay in adhesion maturation. Consequently, the requirement for Piezo1 activity can be bypassed by lowering contractility or elevating membrane tension. In vivo , Piezo1 function in adhesion integrity becomes essential only in aged mice where alterations in tissue mechanics lead to impaired TJs and barrier dysfunction. Collectively these studies reveal an essential function of Piezo1 in the timely establishment and maintenance of cell-cell junctions in the context of a mechanically tensed epidermis.

Article activity feed

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    This manuscript addresses the important topic of cell-cell junction maturation and mechanical stability, with a specific focus on how mechanotransduction through the Piezo1 channel regulates these processes. The authors present compelling in vivo evidence demonstrating that Piezo1 plays a role in junction stability and barrier function, particularly in aged tissue. The work makes a valuable contribution to our understanding of mechanotransduction in epithelial biology. However, several aspects of the mechanistic model and in vitro experiments require additional development to fully support the authors' conclusions.

    Major Strengths:

    • The in vivo experiments are well-designed and provide convincing evidence for Piezo1's role in barrier function
    • The study identifies an important connection between mechanical sensing and junction maturation
    • The age-dependent phenotype provides interesting insights into tissue mechanics
    1. Areas Requiring Additional Development:

    a. Mechanistic Model Definition A major issue is that the central concept of Piezo1 "balancing membrane and cortical tension" requires more precise definition and experimental support. The authors need to clearly explain what this balance means mechanistically and how it is achieved.

    b. Localization-Function Discrepancy There is an important inconsistency between the authors' claims about Piezo1's role and its localization: while they conclude that Piezo1 is crucial for mechanical stability, they also show that Piezo1 is not localized at mature junctions. This apparent contradiction needs to be addressed with a clear mechanistic explanation.

    c. Quantification and Statistical Analysis Several key conclusions would benefit from more rigorous quantification: - The quantitation of junction maturation in Fig. 1a and 2a should include independent analysis of each experiment rather than pooling cells from multiple experiments - Actin morphology and pMLC2 levels at junctions in Fig. 1 need systematic quantification - Cytoskeletal dynamics and morphological changes in Piezo1-eKO cells (Fig. 2a) require quantification

    d. Methodological and Timeline Clarity The analysis methods and temporal aspects of several experiments need better documentation: Analysis Methods:

    The quantification method for mature adhesions (used in Figs. 1a, 1e, 1f, 2a) needs clarification. The Methods section states that "The transition from zipper-like adhesions to mature continuous intercellular junctions were quantified manually," but crucial details are missing:

    • What specific criteria defined a "continuous junction"?
    • Was this based on complete visibility of the cell perimeter as one junction?
    • How were cells classified as having continuous versus zipper-like adhesions?

    e. The protein intensity quantification at junctions requires methodological clarification. The Methods state "For quantifying intensities at junctions, max projection images were generated, and region of interests (ROIs) were restricted to ZO1-positive junctions." However:

    • Were ROIs drawn empirically by the user? Or was the ZO-1 signal used to make a mask?
    • Was there an automated step to determine junctional areas (e.g., intensity threshold)?
    • Was the analysis blinded? If subjective methods were used, this should be clearly stated and potential variability addressed.
    1. Timeline Documentation:

    For blebbistatin experiments (Fig. 1e), specify observation timeframes and quantify the extent of accelerated maturation

    The hypotonic shock experiment (Fig. 3e) timeline needs clarification:

    • When were measurements taken relative to Ca2+ switch?
    • Duration of hypotonic media exposure?
    • Were there time-dependent effects in cell response?
    1. Data Support and Interpretation

    a. Several conclusions require additional support or clarification: - The claim about "more dynamic cytoskeletal motion and irregularly shaped" cells (Fig. 2a) is not supported by the provided data. Quantification of dynamics and cell shape are needed to support this conclusion. Cytoskeletal imaging data would also be useful.

    b. The interpretation of junctional tension requires revision:

    • Current conclusions about increased junctional tension are inferred indirectly from vinculin (Fig. 1c) and a18-catenin (Fig. S1a) immunostaining images.

    • Consider either:

      a) Adding direct junctional tension measurements (e.g., optical measurements, PMID 31964776)

      b) Limiting claims to well-supported morphological differences and moving tension-related interpretations to the Discussion as speculative elements

    c. The description "Analysis of vinculin translocation to intercellular junctions showed reduced levels of vinculin at cell-cell contacts, but abundant vinculin at cell-matrix adhesions (Supplementary Fig. S2a), indicating abnormal build-up of stresses at intercellular junctions of Piezo1-eKO cells" needs revision:

    • "Build-up" suggests higher tensions in Piezo1-eKO cells, which contradicts impaired adhesion maturation findings. Suggest replacing with "distribution" or "organization" "Intercellular" is used ambiguously to include both cell-cell and cell-matrix adhesions
    1. Literature Context:

    The discussion should incorporate recent relevant literature on Piezo1's role in tight junction regulation (e.g., PMID 37005489, PMID 33636174, PMID 31409093)

    1. Technical Considerations
      • For localization studies (Fig. 2), using keratinocytes from Piezo1-tdTomato mouse (JAX #029214) would be preferable to heterologously-expressed Piezo1-FLAG, as it would avoid potential artifacts from non-physiological expression levels
      • Supp Fig. 1b requires additional replicates
      • The Fig. 3A legend states "Note increase in FLIPPER-TR lifetime indicative of elevated membrane tension in Piezo1-eKO" when the data actually shows the opposite - a decrease in Flipper-TR lifetime indicating lower membrane tension
    2. Conceptual and Experimental Clarity Needed Several statements require clearer explanation or additional supporting evidence:

    a. Regarding junction maturation mechanisms:

    The authors state: "This indicated that formation of belt-like adhesions was associated with initial contractility build-up by actomyosin stress fibers linked to junctions, followed by a switch to parallel actomyosin bundles and reduced contractility at adhesions, while the junctions themselves were stabilized in a stressed state indicated by a strengthened actin-junction link." Each part of this claim needs experimental support:

    • The "initial contractility build-up by actomyosin stress fibers linked to junctions" needs to be demonstrated
    • The "switch to parallel actomyosin bundles and reduced contractility at adhesions" requires quantification
    • The claim about "junctions themselves were stabilized in a stressed state" needs stronger evidence

    b. The statement "contact expansion from zippers to a belt requires collaborative regulation of adhesion tension and actomyosin cytoskeleton to lower interfacial tension at the contact" is unclear and needs clarification

    c. The claim "Concomitant with emergence of continuous junctions (8h), the stress fibers were replaced by thick actin bundles positioned perpendicularly to junctions (Fig. 1b)" is not clearly supported by the data

    1. Regarding experimental interpretation:
      • In Fig. 1e, the authors claim that 5µM blebbistatin accelerates junction maturation, but this conclusion is not supported by the statistics (p = 0.0784). Additionally, the timeframe of observation and the quantification of maturation speed should be specified
      • The results section describing Fig. 3 presents seemingly disconnected observations without clear mechanistic links between them, making it difficult to follow the authors' logic and support their conclusions
      • The mechanism by which both reduced contractility (blebbistatin) and increased membrane tension can accelerate maturation (Fig. 1e, f; and also in Piezo1-eKO Fig. 3d, e) needs explanation. The fact that these interventions also accelerate maturation also in Piezo1-eKO suggests a mechanism independent of Piezo1 which is at odds with their broad conclusion that Piezo1 balances membrane tension and cortical contractility in the maturation process. The precise mechanism of Piezo1's role in sensing membrane and cortex tension requires clarification.
      • How Piezo1 maintains mechanical stability of mature junctions despite not being localized there needs to be explained
    2. Suggested Additional Experiments:

    a. Optional: Given the age-dependent tissue stiffness effects proposed by the authors, examining keratinocyte behavior in vitro on substrates of varying stiffness would provide valuable insights

    b. Optional: Direct measurements of tension at cell-cell junctions where Piezo1 localizes would help validate the proposed mechanical model

    1. Minor Points:
      • The cell biology sections, particularly descriptions of in vitro experiments, would benefit from a thorough revision to improve precision and clarity. For instance, the Results section describes "Analysis of vinculin translocation to intercellular junctions" when no translocation is actually being studied
      • Figure legends should clearly indicate what individual data points represent
      • Several conclusions are overstated. For example, the authors conclude that "Piezo1 controls the maturation process" and that "Piezo1 is required for cell junction maturation into junctional belts" based on Fig. 2. These are exaggerated claims since maturation still progresses in Piezo1's absence, just more slowly. "Regulates" or "modulates" would be more appropriate terminology

    In conclusion, while this manuscript presents important findings regarding Piezo1's role in junction maturation and stability, addressing the mechanistic and quantification issues outlined above is essential for supporting the authors' conclusions. The authors have laid groundwork for understanding an important biological process, and addressing these points would help readers better appreciate the significance of their findings.

    Significance

    General Assessment: This study investigates the critical role of mechanosensing in epithelial barrier formation and maintenance, with a particular focus on Piezo1's contribution to junction maturation and stability. The work's primary strengths lie in its compelling in vivo demonstrations of Piezo1's importance for barrier function, particularly in aged tissue, and its identification of a novel connection between mechanical sensing and junction maturation. The age-dependent phenotype provides valuable insights into tissue mechanics and barrier maintenance. However, the mechanistic understanding of how Piezo1 coordinates these processes requires further development, particularly regarding the proposed balance between membrane and cortical tension.

    Advance: This work provides several important advances:

    1. First demonstration of Piezo1's role in regulating the maturation of cell-cell junctions from zipper-like to belt-like structures
    2. Novel insights into how mechanical forces influence junction maturation through mechanosensitive ion channels
    3. Important connection between aging, tissue mechanics, and barrier function
    4. Integration of mechanical sensing with junction assembly and stability

    The findings extend our understanding of epithelial barrier formation beyond traditional molecular pathways to include mechanotransduction, suggesting new therapeutic possibilities for barrier dysfunction. The age-dependent phenotype is particularly significant as it reveals how mechanical properties of tissue influence barrier maintenance over time.

    Audience: This research will be of broad interest to multiple communities:

    • Cell biologists studying junction assembly and epithelial organization
    • Mechanobiologists interested in force transmission and sensing
    • Ion channel researchers interested in the physiological roles of channels
    • Aging researchers investigating tissue barrier function
    • Bioengineers developing therapeutic strategies for epithelial barriers

    The findings have both basic research and translational implications, particularly for understanding and treating age-related barrier dysfunction in epithelia.

    Reviewer Expertise: Cell biology, mechanobiology, live cell imaging, quantitative image analysis, ion channels I have sufficient expertise to evaluate all aspects of the manuscript except for the specific age-related physiological changes in mouse skin, which falls outside my area of expertise.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This manuscript describes the role of the mechanosensitive ion channel Piezo1 in epithelial junction assembly, using Piezo-1-KO primary epidermal keratinocytes in vitro and mouse skin in vivo. The authors conclude that Piezo1 allows balancing of membrane versus cortical tension to stabilize junctions and promote tight juntion (TJ) barrier integrity assembly. The conclusion that Piezo1 has an important function in the formation and maintenance of apical junctions of keratinocytes both in vitro and in vivo is well documented by experiments in WT, KO and rescue cells/tissues where different parameters are carefully measured: protein localization, quantification of mature junctions, membrane tension using the flipper probe, use of the myosin inhibitor blebbistatin, analysis of cortical stiffness by AFM, etc. Although, the physiological relevance and the mechanism through which Piezo operates in young skin are not clear, the authors make reasonable claims, that are not too speculative.

    Major comments:

    1. The Supplementary Figure 4d (panel d) that is described in the Results section is missing. It supposedly shows that 1 year-old Piezo1-eKO mice diplay an increase in transepidermal water loss, inducating that TJ barrier function is compromised. The Figure legend for the panel is also missing. Please provide the Figure panel and the legend.
    2. TJ barrier function depends on claudins, and the loss of claudin-1 leads to transepidermal water loss (please cite the relevant paper from the Tsukita lab). Considering that altered TJ barrier function is observed only in 1-yr old mice (Supplementary Figure to be shown, see point n.1) and not in young mice (Suppl. Fig. 3f-h), the expression pattern of the main claudin isoforms, and especially claudin-1, in the different cell populations (see Suppl. Fig. 3b, or by IF analysis) in young vs old and WT vs KO mice must provided, to provide a mechanistic basis for the observed TJ barrier phenotype. This would help to determine if the phenotype is linked to altered claudin expression or to altered (increased) perijunctional tension.
    3. Mechanistically, the authors mention in the Discussion that Piezo1 might act through RhoA signaling. In Rübsam et al 2017 the authors showed that the uppermost viable layer of the skin has increased apical junctional tension, due to anisotropy of AJ distribution which correlates with EGFR activation and localization. In this context, it is important to know if KO of Piezo-1 affects EGFR localization and signaling, and to probe the RhoA pathway using for example the ROCK inhibitor, instead of blebbistatin.

    Minor comments:

    1. The Methods sections should be improved with additional details. For example, the description of quantification of junctional labeling is vague, and there is often no or little indication in the Legends that specifies number of experiments and junctional segments. In addition, quantification of junctional stainings for specific proteins should be done using a junctional reference marker and not as "absolute" values, because there can be variability of staining between samples and experiments. This is especially important when measuring ZO-1, which is a dual AJ-TJ protein (for example at zipper-like junctions ZO-1 colocalizes with AJ markers). Double labelling with a true TJ marker (occludin or cingulin) and/or a true AJ marker (PLEKHA7, afadin, Ecadherin or a catenin) and quantifying junctional labeling by ratio is highly recommended. This is particularly important when evaluating tension-sensitive epitopes/antigens (alpha-catenin, vinculin, etc)
    2. Please use ZO-1 (and ZO-2) consistently, instead of ZO1 (or ZO2), which is completely inaccurate.
    3. Plase cite Furuse et al 2002 JCB (see above).
    4. Please include statistical data in Figure Legends, specifying the number of separate experiments and number of samples. At least three experiments is recommended.
    5. At the end of the introduction the authors mention "putative" occludin-containing TJs. I would delete putative. Epithelial junctions that contain a continuous circumferential linear distribution of occludin/ZO-1/cingulin and form a barrier comply with the definition of a TJs (Citi et al JCS 2024) .
    6. Please insert page numbers in the manuscript.

    Significance

    The notion that mechanosensitive calcium channels contribute to the formation of continuous apical junctions (repair and assembly) was introduced by the Miller lab, using Xenopus oocytes. This manuscript provides a significant conceptual advance, not only by using in vitro and in vivo mouse (mammalian) epidermal keratinocytes as model system, but especially by using Piezo1-KO and rescue experiments, which was not done in the Xenopus model.

    This research would be of great interest to cell biologists interested in epithelial differentiation, polarization and junction assembly, and to clinicians that are interested in the molecular basis of skin pathophysiology.

    My expertise is in the biochemistry, cell biology and mechanobiology of epithelial junctions. I have used Xenopus embryos, cultured epithelial cells, primary keratinocytes and keratinocyte cell lines and KO mice as model systems. The research of my group focuses on how specific cytoskeletal proteins are organized to transmit forces and are recruited to junctions, and how junctional proteins respond to mechanical force. I have experience in all of the methods described in this paper, except for transepidermal water loss measurement, in situ RNA hybridization and mechanical stretching experiments.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The studies described in this manuscript investigated the mechanical regulation of tight junction (TJ) maturation in the epidermis using a combination of in vitro and in vivo analysis. The findings indicate that during calcium-induced cell-cell adhesion in keratinocytes, there is an initial build up cortical tension in the actin cytoskeleton, followed by an increase in membrane tension, which is required for formation of mature TJs. The studies also demonstrate that loss of Piezo1 delays TJ maturation via defects in membrane tension. Loss of Piezo1 also impaired epidermal homeostasis and barrier function in aged mice. The authors propose that the balance in forces between the cortex and membrane is essential for TJ assembly and is mediated by Piezo1.

    Overall, the studies are carefully designed and executed and provide a clear role for membrane tension and Piezo1 in TJ development, making use of molecular forces sensors, imaging, and chemical and genetic perturbations. However, not all of the conclusions are fully supported by the data, and some key findings require additional quantitative and statistical analysis.

    1. The statement at the end of page 5 ("This indicated that formation of belt-like...) is somewhat overinterpreted from the data shown. To draw conclusions about a switch to reduced contractility at adhesions requires more careful spatio-temporal quantification of F-actin and pmyosin beyond the example single cells shown in 1b. It would also help to see the localization of Ecadherin during this process.
    2. To avoid confusion, the authors should pay careful attention to terminology and be specific when referring to adherens junctions or TJs, rather than just junctions generally.
    3. The labelling of Figure 2b could be clearer. Were the CNL cells also transfected with Piezo1 or mock transfected to control for general effects of transfection? This was not clear from the figure captions.
    4. In Figure 2c-g it is not specified which timepoints the images represent, and the qualitative description of changes in localisation require quantification.
    5. The importance of Piezo1 in junction maturation is somewhat overstated throughout. While Piezo KO clearly delays TJ maturation, the process can still be completed. In the absence of Piezo1 what triggers the rise in membrane tension? Could there be any compensation from Piezo2?
    6. Some of the differences noted are subtle and not strongly significant, such as K6expression, Ca++ induced Piezo1 expression, and F4/80 staining. The conclusions related to these responses should be tempered or qualified.
    7. Analysis of the immune infiltration and the suggested inflammatory response in aged mice is fairly preliminary and not well supported by the data. A second marker of macrophages and addition of T cell markers would help clarify the type of immune response. It would also help to describe the localisation of specific immune cells in more detail and include a direct marker of inflammation (e.g. inflammatory cytokines).
    8. OPTIONAL: Although not essential for the conclusions of the study, the impact and insight could be improved by providing more analysis of the mechanism for the role of Piezo1. For example, does the build up of cortical tension trigger changes in ion channel signalling, and how does this then regulate membrane tension? Is RhoA or aPKC involved?

    Significance

    The process by which epithelia assemble and maintain effective barriers is complex and requires precise spatio-temporal regulation. This study provides some new insight into the mechanical regulation of TJ assembly within the epidermis. It builds upon previous work that identified essential biomechanical cross-talk between adherens junctions and TJs and adds some new information on the timings and specific roles of membrane tension and Piezo1. The interplay between cortical and membrane tension is noteworthy, and this mechanism may have important implications in other barrier tissues. A limitation of the study is a lack of mechanistic detail in how the mechanical switch occurs during TJ maturation, including the specific molecules, structures, and interactions with Piezo1.

    The study also describes the functional implications, whereby loss of Piezo1 in the mouse disrupts barrier integrity. However, these effects were quite subtle. Barrier homeostasis was only disrupted in aged mice, and in vitro, loss of Piezo1 delayed but did not prevent junction maturation. It is therefore interesting to speculate what other mechanisms may be involved in TJ maturation. A potential limitation here is also a lack of detail in the analysis of the inflammatory and immune response in Piezo KO skin.

  5. Version published to 10.1101/2024.11.20.624378v1 on bioRxiv