Piezo1 mechanosensing regulates integrin-dependent chemotactic migration in human T cells
- IICB-Translational Research Unit of Excellence, CSIR-Indian Institute of Chemical Biology, India
- Department of Biological Sciences, Indian Institute of Science Education and Research, India
- Department of Biological Sciences, Indian Association for Cultivation of Science, India
- Academy of Scientific and Innovative Research, India
Figures
Figure 1 with 4 supplements
Piezo1 deficiency abrogates integrin-dependent motility in human T cells which is mediated through redistribution of Piezo1 at the leading edge in response to chemokine stimulation.
(A) MSD versus time calculated for GFP+ (potential Piezo1-knockdown cells) and GFP- (potential control cells) in GFP plasmid and Piezo1 siRNA co-transfected human CD4+ T lymphocytes, that were …
Figure 1—figure supplement 1
Piezo1 facilitates integrin-dependent human CD4+ T lymphocyte migration by localizing to the leading edge of cells in response to chemokine stimulation.
(A) Quantitative PCR analysis of Piezo1 and Piezo2 relative expression, in control and Piezo1 siRNA-transfected CD4+ T lymphocytes. (B) Flow cytometric measurement of Piezo1 mean fluorescence …
Figure 1—animation 1
Representative animation of cell trace-stained CD4+ T lymphocytes transfected with GFP construct and Piezo1 siRNA, in the presence of recombinant human CCL19 on ICAM1-coated plates, for a total duration 15 min at 30 s per frame.
The left panel represents superimposed cell trace and GFP signals of moving cells. The right panel shows tracks of the moving cells. Boxed cells are GFP+ cells. 20X Magnification. Related to Figure …
Figure 1—animation 2
Representative 3D projection of Z-stacks of CD4+ T lymphocyte treated with recombinant human CCL19 for 15 min, immunostained with Piezo1 antibody and Hoechst.
63X magnification. Related to Figure 1F.
Figure 1—animation 3
Representative immunofluorescence time-lapse animation of Piezo1-mCherry expressing Jurkat cell moving on ICAM1-coated dish in the presence of recombinant human SDF1α.
Images were acquired manually for a total duration of 15 min at 1 min per frame interval. Images were captured at 40X magnification. Related to Figure 1l.
Figure 2 with 1 supplement
Piezo1 redistribution in migrating T cells follows increased membrane tension in the leading edge.
(A) Schematic illustration of interference reflection microscopy imaging setup. (B) Probability distribution of the amplitude of temporal fluctuations (SDtime, left) and tension (right) before and …
Figure 2—figure supplement 1
Chemokine stimulation increases leading edge membrane tension in human CD4+ T lymphocytes.
(A) Probability distribution of temporal fluctuation (left) and tension (right) followed over time, (B) Tension maps showing high-tension edges aligning with direction of cell migration (arrows) …
Figure 3 with 2 supplements
Focal adhesion following chemokine receptor activation does not depend on Piezo1.
(A) Representative confocal images of human CD4+ T lymphocytes, fixed and stained for phospho-FAK (pFAK) under untreated and CCL19-treated conditions. (B) Increased polarity of pFAK upon chemokine …
Figure 3—figure supplement 1
Piezo1 acts downstream of focal adhesion formation and localizes to sites of focal adhesions formed in response to chemokine stimulation.
(A) Mean signal intensity of pFAK in CCL19-treated control and Piezo1 KD cells (n>440 random cells, each). (B) Mean signal intensity of paxillin in CCL19-treated control and Piezo1 KD cells (n>780 …
Figure 3—animation 1
Representative 3D rendering of confocal Z-stacks of CD4+ T lymphocyte treated with recombinant human CCL19 for 10 min, immunostained with phosphorylated focal adhesion kinase (pFAK) antibody and Hoechst.
63X magnification. Related to Figure 3A.
Figure 4 with 5 supplements
Membrane recruitment of LFA1 on chemokine receptor activation disrupts Piezo1 deficiency.
(A) Representative confocal images of fixed, immunostained Piezo1 and LFA1 in unstimulated and CCL19-stimulated CD4+ T lymphocytes. (B) Increased LFA1 polarity in response to recombinant CCL19 …
Figure 4—animation 1
Representative 3D rendering of confocal Z-stacks of CD4+ T lymphocyte depicting Piezo1 and CD11a/LFA-1 distribution, upon 30 min of recombinant CCL19 treatment.
63X magnification. Related to Figure 4A.
Figure 4—animation 2
Representative 3D rendering of confocal Z-stacks of control siRNA-transfected CD4+ T lymphocyte depicting CD11a/LFA-1 polarity, upon 30 min of recombinant CCL19 treatment.
63X magnification. Related to Figure 4E.
Figure 4—animation 3
Representative 3D rendering of confocal Z-stacks of Piezo1 siRNA-transfected CD4+ T lymphocyte depicting CD11a/LFA-1 polarity, upon 30 min of recombinant CCL19 treatment.
63X magnification. Related to Figure 4E.
Figure 4—animation 4
Representative 3D rendering of confocal Z-stacks of control siRNA-transfected CD4+ T lymphocyte depicting phosphorylated Akt polarity, upon 30 min of recombinant CCL19 treatment.
63X magnification. Related to Figure 4F.
Figure 4—animation 5
Representative 3D rendering of confocal Z-stacks of Piezo1 siRNA-transfected CD4+ T lymphocyte depicting phosphorylated Akt polarity, upon 30 min of recombinant CCL19 treatment.
63X magnification. Related to Figure 4F.
Figure 5 with 1 supplement
Local Ca2+ mobilization with Piezo1 redistribution upon chemokine stimulation.
(A) Representative total internal reflection fluorescence (TIRF) image of a Jurkat Cell stained with Fluo-3, AM and transiently expressing with Piezo1 mCherry. Lower panel: zoomed-in image and …
Figure 5—figure supplement 1
Active calcium mobilization indicating Piezo1 activity at Piezo1-rich areas of focal adhesions formed independently of Piezo1-mediated actin polymerization.
(A) Scatter plot of how percentage area of Ca2+ peaks (real and simulated) that overlap with Piezo1 varies with area fraction of Piezo1, showing the comparison between real and simulated data. (B) …
Figure 6 with 5 supplements
Piezo1 deficiency disrupts F-actin retrograde flow in T cells despite chemokine receptor activation.
(A) Representative fixed confocal images of actin and Piezo1 distribution in CD4+ T lymphocytes after 30 min of 0.5 µg/ml recombinant CCL19 treatment. (B) Representative fixed confocal images of …
Figure 6—figure supplement 1
Leading edge polarity of Piezo1-mCherry and dynamic distribution of actin-GFP in SDF1α-treated Jurkat cells.
(A) Representative 2D confocal image of Jurkat cell expressing Piezo1 mCherry and actin GFP in the absence (top panel) or presence (bottom panel) of recombinant SDF1α. (B) Box plots depicting Piezo1 …
Figure 6—animation 1
Representative 3D rendering of confocal Z-stacks of CD4+ T lymphocyte depicting Piezo1 and actin distribution, upon 30 min of recombinant CCL19 treatment.
63X magnification. Related to Figure 6A.
Figure 6—animation 2
Representative 3D rendering of confocal Z-stacks of control siRNA-transfected CD4+ T lymphocyte depicting actin polarity, upon 30 min of recombinant CCL19 treatment.
63X magnification. Related to Figure 6B.
Figure 6—animation 3
Representative 3D rendering of confocal Z-stacks of Piezo1 siRNA-transfected CD4+ T lymphocyte depicting loss of actin polarity, after 30 min of recombinant CCL19 treatment.
63X magnification. Related to Figure 6B.
Figure 6—animation 4
Representative time-lapse animation of Piezo1-mCherry/actin-GFP expressing Jurkat cell, moving on ICAM1-coated dish in the presence of recombinant human SDF1α.
Images were acquired as a Z-stack (1µm per stack) at 63X/1.40 oil magnification. Total duration of time-lapse was 5 min at 30 s per frame. Images are represented as maximum intensity projections of …
Figure 7
The mechanistic model depicting the involvement of Piezo1 mechanosensing in leading-edge events in a migrating T cell.
Proposed model suggests chemokine receptor activation in human T cells leads to focal adhesion kinase activation and focal adhesion formation. Focal adhesions lead to localized increase in membrane …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Transfected construct (Homo sapiens) | Piezo1 mCherry; Piezo1-GFP plasmids | From Dr. Charles Cox at Victor Chang Cardiac Research Laboratory, Darlinghurst, Australia (Cox et al., 2016) | ||
| Transfected construct (Homo sapiens) | pCAG-mGFP-Actin plasmid | Addgene | Plasmid #21948, RRID:Addgene_21948 | |
| Antibody | Piezo1, Rabbit, polyclonal | Proteintech | Cat# 15939–1-AP, RRID:AB_2231460 | 5 µg/ml |
| Antibody | Paxillin, Mouse, Clone 5 H11 | Merck, Sigma Aldrich | Cat# 05–417, RRID:AB_309724 | 5 µg/ml |
| Antibody | phospho-FAK (Tyr 397), Rabbit, polyclonal | Santa Cruz | Cat# sc-11765-R, RRID:AB_653198 | 5 µg/ml |
| Antibody | phospho-Akt (Ser 473), Rabbit, polyclonal | Cell Signalling Technolgies | Cat# 9271, RRID:AB_329825 | 5 µg/ml |
| Antibody | Akt1, Goat, polyclonal | Santa Cruz | Cat# sc-1618, RRID:AB_630849 | 5 µg/ml |
| Antibody | anti-human APC CD44, Mouse, Clone G44-26 | BD Bioscience | Cat# 560890, RRID: AB_398683 | 5 µg/ml |
| Antibody | anti-human FITC CD11a/LFA-1, Mouse, Clone G43-25B | BD Pharmingen | Cat# 555379, RRID:AB_395780 | 5 µg/ml |
| Antibody | anti-mouse IgG Alexa Fluor488, Goat | Thermo Fisher, Invitrogen | Cat# A-11029, RRID:AB_2534088 | 1: 350 dilution |
| Antibody | anti-rabbit IgG Alexa Fluor 568, Goat | Thermo Fisher, Invitrogen | Cat# A-11036, RRID:AB_10563566 | 1: 350 dilution |
| Antibody | anti-goat IgG Alexa Fluor 647, Chicken | Thermo Fisher, Invitrogen | Cat# A-21469, RRID:AB_2535872 | 1: 350 dilution |
| Antibody | Human CD4 microbeads, T cells | Miltenyi Biotec | Cat# 130-097-048 | 1: 350 dilution |
| Peptide, recombinant protein | LifeAct Tag-GFP2 protein | Ibidi | Cat# 60112 | 0.5 µg/ml |
| Peptide, recombinant protein | Recombinant Human MIP-3β (CCL19) | Peprotech | Cat# 300-29B | 0.5 µg/ml |
| Peptide, recombinant protein | Recombinant Human ICAM-1 | Peprotech | Cat# 150–05 | 4 µg/ml |
| Peptide, recombinant protein | Recombinant Human SDF-1α (CXCL12) | Peprotech | Cat# 300–28 A | 0.1 µg/ml |
| Peptide, recombinant protein | Recombinant human fibronectin (RetroNectin) | Takara | Cat# T202 | 10 µg/ml |
| Chemical compound, drug | Calpain Inhibitor, PD 150606 | Merck, Sigma Aldrich | Cat# D5946 | 100 µM |
| Chemical compound, drug | FAK Inhibitor 14 | Merck, Sigma Aldrich | Cat# SML0837 | 10 µM |
| Peptide, recombinant protein | Piezo1 Inhibitor, GsMTx4 | Tocris | Cat# 4912 | 10 µM |
| Commercial assay or kit | Lonza P3 Primary Cell 4D-Nucleofector X Kit L | Lonza | Cat# V4XP-3024 | |
| Sequence-based reagent | Human Piezo1 Forward primer | IDT | 5’-CCC AAG TGG AGC TCA GGC CC-3’ | |
| Sequence-based reagent | Human Piezo1 Reverse primer | IDT | 5’-GGG CCA GGG ACA GGC AGA AG-3’ | |
| Sequence-based reagent | Human Piezo2 Forward primer | IDT | 5'-CAT CTA CAG ACT GGC CCA CCC G-3' | |
| Sequence-based reagent | Human Piezo2 Reverse primer | IDT | 5'-AGA GCA CAG TGA GGC GGT CA-3' | |
| Sequence-based reagent | Human 18 S primer | IDT | 5'-GTA ACC CGT TGA ACC CCA TT-3' | |
| Sequence-based reagent | Human 18 S primer | IDT | 5'-CCA TCC AAT CGG TAG TAG CG-3' | |
| Sequence-based reagent | EGFP control mission esiRNA | Merck, Sigma Aldrich | Cat# EHUEGFP | |
| Sequence-based reagent | Human Piezo1 mission esiRNA | Merck, Sigma Aldrich | Cat# EHU135531 | |
| Cell line (Homo sapiens) | Jurkat cell line | From Dr. Santusabuj Das at National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India. ATCC | TIB-152 | |
| Other | Fluo-3, AM (Calcium indicator) | Thermo Fisher, Invitrogen | Cat# F1241 | |
| Other | Hoechst 33342 | Merck, Sigma Aldrich | Cat# 14533 | |
| Other | Cell Trace Violet | Thermo Fisher, Invitrogen | Cat# C34557 | |
| Other | Cell Trace CFSE | Thermo Fisher, Invitrogen | Cat# C34554 | |
| Other | Alexa Fluor 532 Phalloidin | Thermo Fisher, Invitrogen | Cat# A22282 | |
| Software, algorithm | Fiji | Open Source | https://fiji.sc/ | |
| Software, algorithm | MATLAB 2020b | IISER, Kolkata academic license |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/91903/elife-91903-mdarchecklist1-v1.docx
-
Source data 1
Source data ile 1 is an Excel file containing all data corresponding to each figure mentioned in the study.
- https://cdn.elifesciences.org/articles/91903/elife-91903-data1-v1.xlsx
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Source code 1
Source code file 1 includes a zip file containing codes used in Fiji (.ijm files) and MATLAB (.m files) image analysis.
- https://cdn.elifesciences.org/articles/91903/elife-91903-code1-v1.zip
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Supplementary file 1
Representative analyses for calculation of cell polarity of (A) Piezo1 (fixed); (B) Piezo1 mCherry (live); (C) phosphorylated FAK (pFAK, fixed cell); (D) paxillin (fixed cell); (E) CD11a/LFA1 (fixed cell); (F) actin (fixed cell).
Region-of-interest identifying cell boundary and polarity indices mentioned. All analyses were performed on Fiji (see methods).
- https://cdn.elifesciences.org/articles/91903/elife-91903-supp1-v1.zip
