Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing

Keratinocytes, the predominant cell type of the epidermis, migrate to reinstate the epithelial barrier during wound healing. Mechanical cues are known to regulate keratinocyte re-epithelialization and wound healing; however, the underlying molecular transducers and biophysical mechanisms remain elusive. Here, we show through molecular, cellular, and organismal studies that the mechanically activated ion channel PIEZO1 regulates keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls. By imaging the spatiotemporal localization dynamics of endogenous PIEZO1 channels, we find that channel enrichment at some regions of the wound edge induces a localized cellular retraction that slows keratinocyte collective migration. In migrating single keratinocytes, PIEZO1 is enriched at the rear of the cell, where maximal retraction occurs, and we find that chemical activation of PIEZO1 enhances retraction during single as well as collective migration. Our findings uncover novel molecular mechanisms underlying single and collective keratinocyte migration that may suggest a potential pharmacological target for wound treatment. More broadly, we show that nanoscale spatiotemporal dynamics of Piezo1 channels can control tissue-scale events, a finding with implications beyond wound healing to processes as diverse as development, homeostasis, disease, and repair.


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All experiments were independently reproduced as described in the Materials and Methods to reliably support the experimental findings stated in the manuscript. The biological replicate and experiment replication information are detailed in figure legends and Methods section. We define "biological replicates" as the number of mice for in vivo experiments, cells isolated from different pups or litters (in cases where cells from multiple pups of the same genotype were pooled), or cells independently treated with chemical agents.
1 data point was excluded from each of the Control (Piezo1-cKO background; out of 70 total data points), Control (Piezo1-GoF background; out of 40 total data points) (Fig. 1E) and Piezo1-GoF (out of 53 total data points) flicker datasets (Fig. 1H). We identified these data points as being outliers by the Grubb's test. Tracks detected by Kymobutler that were either (a) obvious noise from artifacts in the kymographs or (b) that were located away from the wound edge were removed from any subsequent analyses.

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Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

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• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Comparable keratinocyte cultures were either allocated into groups based on genotypes or randomly assigned into treatment groups (for experiments in the Piezo1-cKO or PIEZO1-tdTomato background). Quantification was performed using analysis pipelines applied to all conditions equally. Blinding was performed during data analysis where appropriate (Flika analysis, single cell migration analyses etc.). During automated analyses, thresholds were chosen based on objective properties and applied across the board.
Custom data analysis code for analyzing data shown in Fig. 5D has been made uploaded as a Source Code file; all other code or software utilized is publicly available and has been cited in the Methods section. The datasets plotted in Figure 1E and 2 have been uploaded as source data files. Source data files for Figure 5C and 5D have been uploaded to Dryad (https://datadryad.org/stash/share/P3SkFE1Nxxs197lcy4HRpY4SG12-DXEoGgPaUeDdaqc).