Research ArticleCIRCADIAN RHYTHM

Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing

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Science Translational Medicine  08 Nov 2017:
Vol. 9, Issue 415, eaal2774
DOI: 10.1126/scitranslmed.aal2774
  • Fig. 1. The cell-intrinsic fibroblast circadian proteome contains numerous cytoskeletal regulators.

    (A) Protein annotation clusters generated by DAVID containing terms enriched (P < 0.10) with rhythmic protein abundances identified by RAIN (Rhythmicity Analysis Incorporating Nonparametric methods) (P < 0.01) from the analysis of primary lung fibroblasts from PER2::LUC mice. (B) The 10 largest gene ontology (GO) (cellular compartment) terms within the rhythmic data set by protein number. (C) Mean abundance [light/heavy (L/H) ratio] of rhythmic proteins from the “actin cytoskeleton” cluster determined by three SILAC experiments with three parallel PER2::LUC measurements indicating the circadian phase (heat map).

  • Fig. 2. CRY-dependent cell-intrinsic rhythms in actin polymerization.

    (A) Schematic depicting rhythms in actin polymerization, which may be cell-intrinsic (blue arrow; driven by circadian gene expression) in addition to systemic cues (red arrow). (B) Immunoblots using anti-actin antibody against fractionated and total protein from PER2::LUC fibroblasts at the indicated times after synchronization in the presence of dimethyl sulfoxide (DMSO) (i) or cytoD (0.5 μM) (ii). F/G-actin ratio is quantified below, with best-fit curves from a comparison of fits (n = 3; mean ± SEM). Three parallel bioluminescence measurements (heat map) are included as a marker for the circadian clock. Because G-actin was in excess, exposures between Western blot panels are not equivalent. (C) Live-cell recordings of actin abundance (SiR-actin) in cells labeled with CellTracker Green (i and ii; scale bar, 100 μm). (iii) SiR-actin intensity over time for eight individual tracks (orange lines) with mean (black) ± SEM (gray) overlayed. (iv) Tracks with a circadian harmonic regression FDR (q value) of <1% and amplitude of >10% of the mean are highlighted and quantified. (D) F/G-actin ratios from wild-type (WT; black line) or cry1−/− cry2−/− (orange line) fibroblasts at the indicated times after synchronization, with best-fit curves from a comparison of fits (n = 3; mean ± SEM); RAIN P values are indicated.

  • Fig. 3. A circadian rhythm in fibroblast wound-healing response.

    (A) (i) Fibroblast monolayers derived from adult PER2::LUC mouse skin were entrained and wounded after 20 to 64 hours in free run. (ii) Images of wound-healing assays; time at wounding is indicated, and residual wound is indicated by the highlighted area (pink) (scale bar, 500 μm). (iii) Quantification of the residual wound after 16 hours of wound healing (line) (n = 4; mean ± SEM) with three parallel PER2::LUC measurements (heat maps). RAIN P value is indicated. (B) (i) Fibroblasts labeled with CellTracker Red healing after wounding at the indicated times after synchronization (t). Scale bars, 100 μm. Wound healing (ii) and leading cell velocity (iii) are quantified (n = 4 or 5; mean ± SEM). P values from Tukey’s multiple comparisons test after 60 hours of healing (tH = 60) (ii) or tH = 0 (iii) are indicated. (C) (i) Fibroblasts labeled with SiR-actin (red) and CellTracker Green (cyan) treated as in (B). Scale bars, 50 μm. A single cell for each condition has been highlighted in white with time healing indicated. The center of mass for each label (ii) was determined over 9 hours of healing, and the mean ± SEM degree of polarization (Δx) is indicated (iii). The P value from a two-way analysis of variance (ANOVA) is indicated.

  • Fig. 4. Actin polymerization rhythms are required for circadian regulation of adhesion and wound-healing efficacy by fibroblasts.

    (A) Impedance measurements from cry1−/− cry2−/− (blue) or wild-type fibroblasts treated with DMSO (black) or cytoD (orange) with simultaneous PER2::LUC measurements (heat maps) (n = 6 to 8; mean ± SEM). AU, arbitrary unit. (B) Quantification of mean fibroblast monolayer healing after wounding at the indicated times after synchronization (t) in the presence of 0.5 μM cytoD or vehicle (n = 6 to 12; mean ± SEM). P values from an ANOVA with Tukey’s test for multiple comparisons are indicated.

  • Fig. 5. Diurnal variation in wound-healing outcome and fibroblast mobilization.

    (A) Bioluminescence recording of PER2 expression in neonatal (P5) skin explants from PER2::LUC mice (n = 6; mean ± SEM). (B) Mouse skin wounds before and after 48 hours of healing. Fibroblasts were identified by anti-vimentin reactivity (red) and morphology and quantified by the number (C) and volume (D) (n = 6 to 7; mean ± SEM); Holm-Sidak’s adjusted P value is indicated. Scale bars, 200 μm. DAPI, 4,6′-diamidino-2-phenylindole. (E) Transverse sections (60 μm) of mouse wounds made during the active and resting phases stained using anti-vimentin (magenta) and Hoechst (blue). (F) Cross-sectional vimentin staining across wound edges was quantified (mean ± SEM). (G) The area under the curve (AUC) was calculated using distal vimentin as a baseline [n = 16 (active) or 20 (resting); mean ± SEM]; The P value from a Student’s t test is indicated.

  • Fig. 6. A circadian rhythm in keratinocyte wound healing and a diurnal variation in human burn healing outcome.

    (A) (i) Synchronized HaCaT monolayers expressing luciferase under control of the BMAL1 promoter (n = 24; mean ± SD) were wounded at the indicated times (vertical lines), and (ii) healing was monitored by confocal microscopy. (iii) Relative fluorescence in the wound area (n = 4; mean ± SEM) was calculated, and (iv) maximal healing after 15 hours was compared by Tukey’s multiple comparisons test (P values are indicated). (B) Mean time to 95% healing ± SEM from 118 human burn incidents separated by time of burn occurrence in 4-hour (left) or 12-hour (right) bins. ANOVA P value is indicated, as is the P value for Welch’s t test comparing daytime versus nighttime wounds. P values from Holm-Sidak’s test versus the 0000 to 0359 bin are indicated below. NA, not applicable.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/415/eaal2774/DC1

    Materials and Methods

    Fig. S1. Cell-intrinsic rhythms in the fibroblast proteome.

    Fig. S2. Validation of cofilin 2 and RCC2 circadian abundance.

    Fig. S3. Circadian clocks are robust against actin-modulating drugs.

    Fig. S4. Cell-intrinsic rhythms in actin polymerization in NIH3T3 cells.

    Fig. S5. Cell division in healing fibroblast monolayers.

    Fig. S6. Cell area, velocity, and cortical actin distribution are not circadian in confluent cultures with circadian PER2::LUC activity.

    Fig. S7. Circadian rhythms in actin dynamics and wound-healing efficacy are CRY-dependent in embryonic fibroblasts.

    Fig. S8. Fibroblast monolayer healing in the presence of cytoD.

    Fig. S9. Rho inhibition disrupts impedance rhythms and time-of-wounding effects without disrupting the circadian clock.

    Fig. S10. Collagen deposition and fibroblast distribution after 14 days of healing.

    Fig. S11. A model of clock control of wound healing.

    Fig. S12. Circadian rhythmicity of the “Regulation of actin cytoskeleton” pathway.

    Table S1. DAVID clustering of rhythmic proteins identified by RAIN (P < 0.01).

    Table S2. Individual subject-level data for n < 20 (provided as an Excel file).

    Movie S1. Healing of fibroblast monolayers wounded 24 hours after synchronization.

    Movie S2. Healing of fibroblast monolayers wounded 32 hours after synchronization.

    Movie S3. Fibroblast invasion of wounds made in the active phase after 48 hours of healing.

    Movie S4. Fibroblast invasion of wounds made in the inactive phase after 48 hours of healing.

    References (4856)

  • Supplementary Material for:

    Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing

    Nathaniel P. Hoyle,* Estere Seinkmane, Marrit Putker, Kevin A. Feeney, Toke P. Krogager, Johanna E. Chesham, Liam K. Bray, Justyn M. Thomas, Ken Dunn, John Blaikley, John S. O'Neill*

    *Corresponding author. Email: nhoyle{at}mrc-lmb.cam.ac.uk (N.P.H.); oneillj{at}mrc-lmb.cam.ac.uk (J.S.O.)

    Published 8 November 2017, Sci. Transl. Med. 9, eaal2774 (2017)
    DOI: 10.1126/scitranslmed.aal2774

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Cell-intrinsic rhythms in the fibroblast proteome.
    • Fig. S2. Validation of cofilin 2 and RCC2 circadian abundance.
    • Fig. S3. Circadian clocks are robust against actin-modulating drugs.
    • Fig. S4. Cell-intrinsic rhythms in actin polymerization in NIH3T3 cells.
    • Fig. S5. Cell division in healing fibroblast monolayers.
    • Fig. S6. Cell area, velocity, and cortical actin distribution are not circadian in confluent cultures with circadian PER2::LUC activity.
    • Fig. S7. Circadian rhythms in actin dynamics and wound-healing efficacy are CRY-dependent in embryonic fibroblasts.
    • Fig. S8. Fibroblast monolayer healing in the presence of cytoD.
    • Fig. S9. Rho inhibition disrupts impedance rhythms and time-of-wounding effects without disrupting the circadian clock.
    • Fig. S10. Collagen deposition and fibroblast distribution after 14 days of healing.
    • Fig. S11. A model of clock control of wound healing.
    • Fig. S12. Circadian rhythmicity of the “Regulation of actin cytoskeleton” pathway.
    • Table S1. DAVID clustering of rhythmic proteins identified by RAIN (P < 0.01).
    • Legends for movies S1 to S4
    • References (4856)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2. Individual subject-level data for n < 20 (provided as an Excel file).
    • Movie S1 (.mov format). Healing of fibroblast monolayers wounded 24 hours after synchronization.
    • Movie S2 (.mov format). Healing of fibroblast monolayers wounded 32 hours after synchronization.
    • Movie S3 (.mov format). Fibroblast invasion of wounds made in the active phase after 48 hours of healing.
    • Movie S4 (.mov format). Fibroblast invasion of wounds made in the inactive phase after 48 hours of healing.

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