Piezo1 mechanosensing regulates integrin-dependent chemotactic migration in human T cells

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.

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.