Piezo1 mechanosensing regulates integrin-dependent chemotactic migration in human T cells
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Curated by eLife
eLife assessment
This study provides useful insights into the subcellular localization, interaction with integrins, and functional importance of the cell surface receptor Piezo1 in migrating human T-cells. Whether Piezo1 is critically sensing mechano-physical cues during T-cell migration is however not well supported by direct experimental evidence. The data collected is solid otherwise.
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- Immunology and Inflammation (eLife)
Abstract
T cells are crucial for efficient antigen-specific immune responses and thus their migration within the body, to inflamed tissues from circulating blood or to secondary lymphoid organs, plays a very critical role. T cell extravasation in inflamed tissues depends on chemotactic cues and interaction between endothelial adhesion molecules and cellular integrins. A migrating T cell is expected to sense diverse external and membrane-intrinsic mechano-physical cues, but molecular mechanisms of such mechanosensing in cell migration are not established. We explored if the professional mechanosensor Piezo1 plays any role during integrin-dependent chemotaxis of human T cells. We found that deficiency of Piezo1 in human T cells interfered with integrin-dependent cellular motility on ICAM-1-coated surface. Piezo1 recruitment at the leading edge of moving T cells is dependent on and follows focal adhesion formation at the leading edge and local increase in membrane tension upon chemokine receptor activation. Piezo1 recruitment and activation, followed by calcium influx and calpain activation, in turn, are crucial for the integrin LFA1 (CD11a/CD18) recruitment at the leading edge of the chemotactic human T cells. Thus, we find that Piezo1 activation in response to local mechanical cues constitutes a membrane-intrinsic component of the ‘outside-in’ signaling in human T cells, migrating in response to chemokines, that mediates integrin recruitment to the leading edge.
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Author Response
The following is the authors’ response to the original reviews.
Response to comments of editor/s:
• With regard to the comments on nonavailability of representative images/videos for Figures 1 A and B, in the revised manuscript we have added a representative video of GFP (-) and GFP (+) tracks in Supplemental video 1.
Response to comments of reviewer 2:
• With respect to the concern on figure 1, we have changed ‘% CD4+ T cell Migration’ to ‘% Proportion CD4+ T cell migration’ in Figures 1D & 1E in the revised manuscript. We also labelled the upper and lower panels of Figure 1I as ‘Untreated’ and ‘SDF1α’ respectively.
Response to comments of reviewer 1:
• With regard to the concern that ‘The transfection alone with siRNA may cause the lack of polarity’, we have added comparison of 2D migration MSD between control …
Author Response
The following is the authors’ response to the original reviews.
Response to comments of editor/s:
• With regard to the comments on nonavailability of representative images/videos for Figures 1 A and B, in the revised manuscript we have added a representative video of GFP (-) and GFP (+) tracks in Supplemental video 1.
Response to comments of reviewer 2:
• With respect to the concern on figure 1, we have changed ‘% CD4+ T cell Migration’ to ‘% Proportion CD4+ T cell migration’ in Figures 1D & 1E in the revised manuscript. We also labelled the upper and lower panels of Figure 1I as ‘Untreated’ and ‘SDF1α’ respectively.
Response to comments of reviewer 1:
• With regard to the concern that ‘The transfection alone with siRNA may cause the lack of polarity’, we have added comparison of 2D migration MSD between control EGFP siRNA and Piezo1 siRNA-transfected CD4+ T cells as Supplementary Figure 1E.
• We have added new references as ref 42 and 43, with respect to PIEZO1 association with focal adhesions.
• With regard to the concerns around co-localization of Piezo1 and focal adhesions, we have added a representative image of Piezo1 and pFAK co-localization upon treatment of chemokine in revised Supplementary Fig. 3C. We have also used an additional focal adhesion marker, paxillin, to show that focal adhesion formation is not affected by Piezo1 KD (Revised Fig. 3E-3H). Upon comparing the mean pFAK and paxillin intensities, we observed no difference in Control and Piezo1 KD CD4+ T cells (Supplementary Figs. 3A, B).
• All the minor concerns and suggestions have been taken care of in the revised manuscript.
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eLife assessment
This study provides useful insights into the subcellular localization, interaction with integrins, and functional importance of the cell surface receptor Piezo1 in migrating human T-cells. Whether Piezo1 is critically sensing mechano-physical cues during T-cell migration is however not well supported by direct experimental evidence. The data collected is solid otherwise.
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Joint Public Review:
This work by Liu CSC et al. is an extension of the author's previous work on the role of Piezo1 mechano-sensor in human T cell activation. In this study, the authors address whether Piezo1 plays a role in T-cell chemotactic migration.
The authors used CD4+ T cells or Jurkat T cells to test the effects of siRNA-mediated depletion of Piezo1 on chemotactic migration. They establish that Piezo1 is implicated in chemotactic migration, although the effects of depletion are relatively moderate.
They show that Piezo1 is redistributed to the leading edge of T-cells.
They identify that relocation of Piezo1 to the leading edge follows an increase in membrane tension.
In Piezo-1 depleted cells, they observe a moderate reduction of LFA-1 polarity. With the use of specific inhibitors, they propose Piezo1 activation to be …
Joint Public Review:
This work by Liu CSC et al. is an extension of the author's previous work on the role of Piezo1 mechano-sensor in human T cell activation. In this study, the authors address whether Piezo1 plays a role in T-cell chemotactic migration.
The authors used CD4+ T cells or Jurkat T cells to test the effects of siRNA-mediated depletion of Piezo1 on chemotactic migration. They establish that Piezo1 is implicated in chemotactic migration, although the effects of depletion are relatively moderate.
They show that Piezo1 is redistributed to the leading edge of T-cells.
They identify that relocation of Piezo1 to the leading edge follows an increase in membrane tension.
In Piezo-1 depleted cells, they observe a moderate reduction of LFA-1 polarity. With the use of specific inhibitors, they propose Piezo1 activation to be downstream of focal adhesion formation and upstream of calpain-mediated LFA-1, integrin alpha L beta 2, or CD11a/CD18 recruitment at the leading edge.
Strengths:
Together with their 2018 paper, this study presents Pieszo1 as a regulator of T-cell activation, implicating it as a player in the coordination of the chemotactic immune response.
Weaknesses:
Most of the effects observed are relatively modest. The authors did not challenge the cells with various physico-mechanical conditions to see when Piezo-1 might become really important. For instance, there are no experiments that expose T cells to varying counter-acting forces to see how piezo1 might affect migration.Technical weaknesses:
The authors state that "these high tension edges are usually further emphasized at later time points", but after ten minutes the median tension and tension (Figure 2C and Supplementary Figure 2C respectively) reduce down to the pretreatment time point. It would be clearer if the author stated within which timeframe the tension edges are "further emphasized".
Figures 3 and 4 - The author states the number of cells quantified from the images, but it is not clear whether the data is actually from 3 biological replicates.
Some of the data has no representative images or videos included. There is no video in the supplementary for Figures 1 A and B. There are no representative images of transwell migration assay in Figures 1 D and E.
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Author Response
We are happy that the novelty and strengths of the study have been appreciated by the editor/s and reviewer/s. We thank the editor/s and reviewer/s for a considerably detailed and constructive review of the manuscript. Here are the responses and proposed revisions from the authors.
• The weakness, as pointed out in the editorial comment regarding the absence of data on role of Piezo1 in migrating T cells in varying physico-chemical conditions were, in the opinion of the authors, beyond the scope of the present manuscript. Moreover, introducing external forces using invasive techniques followed by assessment of Piezo1 function was intentionally avoided. That was the reason for using the non-invasive microscopy technique like IRM to assess membrane tension generation in migrating T cells.
• With regard to the …
Author Response
We are happy that the novelty and strengths of the study have been appreciated by the editor/s and reviewer/s. We thank the editor/s and reviewer/s for a considerably detailed and constructive review of the manuscript. Here are the responses and proposed revisions from the authors.
• The weakness, as pointed out in the editorial comment regarding the absence of data on role of Piezo1 in migrating T cells in varying physico-chemical conditions were, in the opinion of the authors, beyond the scope of the present manuscript. Moreover, introducing external forces using invasive techniques followed by assessment of Piezo1 function was intentionally avoided. That was the reason for using the non-invasive microscopy technique like IRM to assess membrane tension generation in migrating T cells.
• With regard to the explanation sought for the statement 'these high tension edges are usually further emphasized at later time points', the edges are visible right from 1 min (Supp fig 2B) and seen to be emphasized at 30 min. In Fig 2D, we find the 3 min time point at which increased tension at edges is visible together with a clear difference in median tension too. Fig. 2c and Supp fig 2C are averaged over all cells - hence it is possible that at a time point when a particular cell still shows higher tension at edges the median tension of Fig 2C is not significantly different. Also, if only a thin section of cell-edge enhances tension - it may contribute to a second peak without affecting the median much.
• With regard to the query regarding experimental replicates, all data shown is derived from at least 3 experimental replicates for Jurkat cells or independent blood donors for primary CD4+ T lymphocytes as specified in the respective figure legends.
• With regard to the comments on nonavailability of representative images/videos for Figures 1 A and B, in the revised manuscript we will add representative video of GFP (-) and GFP (+) tracks. The transwell experiments were assessed by collecting cells from the bottom chamber followed by flow cytometry. We did not take microscopic images of the bottom chambers before collecting the cells.
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eLife assessment
This study provides useful insights into the subcellular localization, interaction with integrins, and function of the cell surface receptor Piezo1 in migrating human T-cells. The data collected is convincing but incomplete. Therefore the idea that Piezo1 is critically sensing mechano-physical cues during T-cell migration is not well supported by direct experimental evidence.
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Joint Public Review:
This work by Liu CSC et al. is an extension of the author's previous work on the role of Piezo1 mechano-sensor in human T cell activation. In this study, the authors address whether Piezo1 plays a role in T-cell chemotactic migration.
The authors used CD4+ T cells or Jurkat T cells to test the effects of siRNA-mediated depletion of Piezo1 on chemotactic migration. They establish that Piezo1 is implicated in chemotactic migration, although the effects of depletion are relatively moderate.
They show that Piezo1 is redistributed to the leading edge of T-cells.
They identify that relocation of Piezo1 to the leading edge follows an increase in membrane tension.
In Piezo-1 depleted cells, they observe a moderate reduction of LFA-1 polarity. With the use of specific inhibitors, they propose Piezo1 activation to be …
Joint Public Review:
This work by Liu CSC et al. is an extension of the author's previous work on the role of Piezo1 mechano-sensor in human T cell activation. In this study, the authors address whether Piezo1 plays a role in T-cell chemotactic migration.
The authors used CD4+ T cells or Jurkat T cells to test the effects of siRNA-mediated depletion of Piezo1 on chemotactic migration. They establish that Piezo1 is implicated in chemotactic migration, although the effects of depletion are relatively moderate.
They show that Piezo1 is redistributed to the leading edge of T-cells.
They identify that relocation of Piezo1 to the leading edge follows an increase in membrane tension.
In Piezo-1 depleted cells, they observe a moderate reduction of LFA-1 polarity. With the use of specific inhibitors, they propose Piezo1 activation to be downstream of focal adhesion formation and upstream of calpain-mediated LFA-1, integrin alpha L beta 2, or CD11a/CD18 recruitment at the leading edge.
Strengths:
Together with their 2018 paper, this study presents Pieszo1 as a regulator of T-cell activation, implicating it as a player in the coordination of the chemotactic immune response.Weaknesses:
Most of the effects observed are relatively modest. The authors did not challenge the cells with various physico-mechanical conditions to see when Piezo-1 might become really important. For instance, there are no experiments that expose T cells to varying counter-acting forces to see how piezo1 might affect migration.Technical weaknesses:
The authors state that "these high tension edges are usually further emphasized at later time points", but after ten minutes the median tension and tension (Figure 2C and Supplementary Figure 2C respectively) reduce down to the pretreatment time point. It would be clearer if the author stated within which timeframe the tension edges are "further emphasised".Figures 3 and 4 - The author states the number of cells quantified from the images, but it is not clear whether the data is actually from 3 biological replicates.
Some of the data has no representative images or videos included. there is no video in the supplementary for Figures 1 A and B. There are no representative images of transwell migration assay in Figures 1 D and E.
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