Circadian actin dynamics drive rhythmic fibroblast mobilization during wound healing

Abstract

Fibroblasts are primary cellular protagonists of wound healing. They also exhibit circadian timekeeping, which imparts an approximately 24-hour rhythm to their biological function. We interrogated the functional consequences of the cell-autonomous clockwork in fibroblasts using a proteome-wide screen for rhythmically expressed proteins. We observed temporal coordination of actin regulators that drives cell-intrinsic rhythms in actin dynamics. In consequence, the cellular clock modulates the efficiency of actin-dependent processes such as cell migration and adhesion, which ultimately affect the efficacy of wound healing. Accordingly, skin wounds incurred during a mouse's active phase exhibited increased fibroblast invasion in vivo and ex vivo, as well as in cultured fibroblasts and keratinocytes. Our experimental results correlate with the observation that the time of injury significantly affects healing after burns in humans, with daytime wounds healing ~60% faster than nighttime wounds. We suggest that circadian regulation of the cytoskeleton influences wound-healing efficacy from the cellular to the organismal scale.

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 Rhythmicity Analysis Incorporating Nonparametric methods (RAIN) (P<0.01) from analysis of primary lung fibroblasts from PER2::LUC mice. B. The 10 largest Gene Ontology (GO) (cellular compartment) terms within the rhythmic dataset by protein number. C. Mean abundance (Light:Heavy (L:H) ratio) of rhythmic proteins from the ‘actin cytoskeleton’ cluster determined by 3 SILAC experiments with 3 parallel PER2::LUC measurements indicating the circadian phase (heat map).

Fig. 2. CRY-dependent cell-intrinsic rhythms in actin polymerisation

A. Schematic depicting rhythms in actin polymerisation, 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 time after synchronisation in the presence of 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±Standard Error (SEM)). 3 parallel bioluminescent measurements (heat map) are included as a marker for the circadian clock. As G actin was in excess, exposures between western panels are not equivalent. C. Live cell recordings of actin abundance (SiR-actin) in cells labelled with Celltracker Green (i,ii, scale bar =100 μm). iii. SiR-actin intensity over time for 8 individual tracks (orange lines) with mean (black) ±SEM (grey) overlayed. Tracks with a circadian harmonic regression FDR (q value) <1% and amplitude >10% of the mean are highlighted and quantified (iv). D. F:G actin ratios from wild type (WT, black line) or cry1-/- cry2-/- (orange line) fibroblasts at the indicated time after synchronisation, with best-fit curves from a comparison of fits (n=3, mean±SEM, RAIN p-values indicated).

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

A. Fibroblast monolayers derived from adult PER2::LUC mouse skin were entrained and wounded after 20-64 hours in free run (i). (ii) Images of wound healing assays; time at wounding is indicated, residual wound is indicated by pink highlighting (scale bar = 500 μm). (iii) Quantification of the residual wound after 16 hours of wound healing (line, mean±SEM, n=4) with 3 parallel PER2::LUC measurements (heat maps). RAIN p-value indicated. Bi. Fibroblasts labelled with CellTracker Red healing after wounding at the indicated time after synchronisation (t). Scale=100 μm. Wound healing (ii) and leading cell velocity (iii) are quantified (n=4 or 5, ±SEM). P values from Tukey’s multiple comparisons test after 60 hours of healing (t_H60) (ii) or t_H0 (iii) are indicated. Ci Fibroblasts labelled with SiR-actin (red) and CellTracker Green (cyan) treated as in B (scale bar = 50 μm). A single cell for each condition has been highlighted in white with time healing indicated. The centre of mass for each label (ii) was determined over 9 hours of healing and the mean (±SEM) degree of polarisation (Δx) is indicated (iii). The P value from a two-way analysis of variance (ANOVA) is indicated.

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

A. Impedance measurements from cry1^-/-cry2^-/- (blue) or WT fibroblasts treated with DMSO (black) or cytoD (orange) with simultaneous PER2::LUC measurements (heat maps) (mean±SEM, n= 6-8). B. Quantification of mean fibroblast monolayer healing after wounding at the indicated time post-synchronisation (t) in the presence of 0.5 μM cytoD or vehicle (n=6-12, ±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 mobilisation

A. Bioluminescent recording of PER2 expression in neonatal (P5) skin explants from PER2::LUC mice (mean±SEM n=6). B. Mouse skin wounds before and after 48 hours of healing. Fibroblasts were identified by anti-vimentin reactivity (red) and morphology, and quantified by number (C ) and volume (D) (mean±SEM, n=6-7, Holm-Sidak’s adjusted P value is indicated). Scale bar =200 μm. E. 60 μm transverse sections of mouse wounds made during the active and resting phases stained using anti-vimentin (magenta) and Hoescht (blue). Cross-sectional vimentin staining across wound edges was quantified (F, mean±SEM) and Area Under Curve (AUC) was calculated using distal vimentin as a baseline (G) (mean±SEM, n=16 (active) or 20 (resting), P 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. Synchronised human HaCaT keratinocyte monolayers expressing luciferase under control of the BMAL1 promoter (i, mean±SD, n=24) were wounded at the indicated times (vertical lines) and healing monitored by confocal microscopy (ii). Relative fluorescence in the wound area (iii, mean±SEM n=4) was calculated and maximal healing after 15 hrs was compared by Tukey’s multiple comparisons test (iv, P values are indicated) B. Mean time to 95% healing ±SEM from 118 human burn incidents separated by time of burn occurrence in 4 (left) or 12 hour (right) bins. ANOVA P value is indicated, as is the P value for Welch’s t-test comparing daytime vs night-time wounds. P values from Holm-Sidak’s test versus the 0000-0359 bin are indicated below.