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. 2023 Dec 6;24(12):e56870.
doi: 10.15252/embr.202356870. Epub 2023 Nov 16.

Circadian oscillation in primary cilium length by clock genes regulates fibroblast cell migration

Ryota Nakazato  1 , Yuki Matsuda  2 , Faryal Ijaz  1 , Koji Ikegami  1   3
Affiliations

Circadian oscillation in primary cilium length by clock genes regulates fibroblast cell migration

Ryota Nakazato et al. EMBO Rep. .

Abstract

Various mammalian cells have autonomous cellular clocks that are produced by the transcriptional cycle of clock genes. Cellular clocks provide circadian rhythms for cellular functions via transcriptional and cytoskeletal regulation. The vast majority of mammalian cells possess a primary cilium, an organelle protruding from the cell surface. Here, we investigated the little-known relationship between circadian rhythm and primary cilia. The length and number of primary cilia showed circadian dynamics both in vitro and in vivo. The circadian rhythm of primary cilium length was abolished by SR9011 and Bmal1 knockout. A centrosomal protein, pericentrin, transiently accumulates in centriolar satellites, the base of primary cilia at the shortest cilia phase, and induces elongation of primary cilia at the longest cilia phase in the circadian rhythm of primary cilia. In addition, rhythmic cell migration during wound healing depends on the length of primary cilia and affects the rate of wound healing. Our findings demonstrate that the circadian dynamics of primary cilium length by clock genes control fibroblast migration and could provide new insights into chronobiology.

Keywords: cell migration; circadian rhythm; clock genes; pericentrin; primary cilia.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Length of primary cilia in cultured cells exhibits circadian rhythm

  1. Schematic diagram of the protocol for induction of ciliogenesis by serum starvation and synchronization of clock genes expression by exposure of cultured NIH/3T3 cells to dexamethasone (DEX).

  2. Real‐time luciferase bioluminescence assay for Per1 promoter activity levels in Per1::Luc‐expressing NIH/3T3 cells exposed to DEX as in (A).

  3. Immunostaining for primary cilia in NIH/3T3 cells at the indicated time points following treatment with the protocol used in panel (A). Red denotes labeling of cilia by anti‐Arl13b antibody, green denotes labeling of centrosomes by anti‐γ‐tubulin antibody, and blue denotes labeling of DNA by DAPI. White arrows point to primary cilia. Scale bar, 20 μm.

  4. Quantitative analysis of the percentage of Arl13b+ NIH/3T3 cells at each indicated time point. The data are from three independent experiments.

  5. Quantitative analysis of the primary cilium length in NIH/3T3 cells at each indicated time point. The data are from three independent experiments. Representative views of DEX‐synchronized primary cilia are provided on the graph. Scale bar, 2 μm.

Data information: Data in panels D and E are presented as mean ± SEM. *P ≤ 0.05 and **P ≤ 0.01 (one‐way ANOVA). Source data are available online for this figure.
Figure EV1
Figure EV1. Circadian rhythm of primary cilium length is continuous for more than 48 h

  1. Representative time‐lapse images of Arl13b‐venus‐expressing NIH/3T3 cells at the indicated time and quantitative analysis of primary cilium length from Movies EV3 and EV4. Scale bar, 2 μm.

  2. Quantitative analysis of primary cilium length in NIH/3T3 cells treated with 2 μM SR9011 at each indicated time point after release from DEX.

Source data are available online for this figure.
Figure 2
Figure 2. Clock genes contribute to circadian rhythms of primary cilium length

  1. Schematic diagram of molecular core clock mechanism and SR9011 pharmacology.

  2. Immunostaining of primary cilia in NIH/3T3 cells treated with 10 μM SR9011. Scale bar, 5 μm.

  3. Quantitative analysis of length of primary cilia in panel (B). The data are from three independent experiments.

  4. Real‐time qPCR analysis of Bmal1 mRNA expression levels in the NIH/3T3 cells incubated with SR9011 at 30 h after release from DEX. The data are from three independent experiments.

  5. Quantitative analysis of primary cilium length in NIH/3T3 cells treated with the indicated concentration of SR9011 24 or 30 h after release from DEX. The data are from over 50 cells per condition.

  6. Immunostaining for primary cilia in NIH/3T3 cells treated with 2 μM SR9011 30 or 42 h after release from DEX. Scale bar, 5 μm.

  7. Quantitative analysis of primary cilium length in NIH/3T3 cells at each indicated time point after release from DEX in panel (F). The data are from three independent experiments.

  8. Immunostaining for primary cilia in WT or Bmal1‐KO NIH/3T3 cells at indicated time point after release from DEX. Scale bar, 5 μm.

  9. Quantitative analysis of primary cilium length in WT or Bmal1‐KO NIH/3T3 cells at each indicated time point after release from DEX in panel (H). The data are from three independent experiments.

Data information: Data in panels C, D, G, and I are presented as mean ± SEM. *P ≤ 0.05 and **P ≤ 0.01 (one‐way ANOVA). Source data are available online for this figure.
Figure 3
Figure 3. Length of primary cilia also exhibits circadian rhythm in murine brain

  1. Immunostaining for primary cilia on neurons (green; labeled by anti‐AC3 antibody) and neural markers (red; labeled with anti‐NeuN antibody) or primary cilia on astrocytes (green; labeled by anti‐Arl13b antibody) and astroglial markers (red; labeled by anti‐GFAP antibody) in murine brain hippocampus. Scale bar, 20 μm.

  2. Immunostaining for primary cilia in neurons in the hippocampus of perfusion‐fixed murine brains at ZT5 or ZT17. Scale bar, 20 μm.

  3. Quantitative analysis of primary cilium length in panel B (n = 31 cilia per condition).

  4. Immunostaining for primary cilia on astrocytes in the hippocampus of perfusion‐fixed murine brains at ZT5 or ZT17. Scale bar, 5 μm. White arrows point to primary cilia.

  5. Quantitative analysis of primary cilium length from panel D (n = 48–53 cilia per condition).

Source data are available online for this figure.
Figure 4
Figure 4. Pericentrin accumulates around the base of shortest primary cilia

  1. Immunostaining for primary cilia and pericentrin. Arl13b is stained red, pericentrin (PCNT) green, and DNA blue (by DAPI) in NIH/3T3 cells at 30 and 42 h after release from DEX. Scale bar, 5 μm. White dotted lines enclose PCNT‐positive granules.

  2. Quantitative analysis of areas positive for PCNT in NIH/3T3 cells treated with SR9011 or dimethyl sulfoxide (DMSO) as vehicle at the indicated time points after release from DEX. The data are from three independent experiments.

  3. Western blot analyses of expression of PCNT and GAPDH (as an internal standard) in NIH/3T3 cells at each indicated time point after release from DEX.

  4. Quantitative analysis of puncta density for PCNT in NIH/3T3 cells treated with SR9011 or vehicle at 30 h after release from DEX (n = 24 cells per condition).

  5. Immunostaining for PCM1 in NIH/3T3 cells treated with SR9011 or vehicle at indicated time points after release from DEX. Scale bar, 5 μm.

  6. Quantitative analysis of areas positive for PCM1 in NIH/3T3 cells treated with SR9011 or vehicle at the indicated time points after release from DEX. The data are from three independent experiments.

Data information: In panels B and F, data are presented as mean ± SEM. *P ≤ 0.05 (one‐way ANOVA). Source data are available online for this figure.
Figure 5
Figure 5. The effect of PCNT‐positive centriolar satellites on circadian rhythm of primary cilium length

  1. Immunostaining for microtubules (green; labeled with anti‐α‐tubulin antibody) and DNA (blue; DAPI) in NIH/3T3 cells incubated with 200 nM colchicine or DMSO as vehicle for 2 h. Scale bar, 10 μm.

  2. Immunostaining for primary cilia (red, Arl13b; green, PCNT) in NIH/3T3 cells treated with 200 nM colchicine or DMSO 30 h after release from DEX. White dotted lines enclose PCNT‐positive granules. Scale bar, 20 μm.

  3. Quantitative analysis of the areas positive for PCNT in NIH/3T3 cells with the indicated concentration of colchicine 30 h after release from DEX. The data are from three independent experiments.

  4. Schematic diagram of the protocol for NIH/3T3 cells synchronized with DEX and exposure to colchicine.

  5. Immunostaining of primary cilia (red; Arl13b) and centrosomes (green; γ‐tubulin) in NIH/3T3 cells treated with 200 nM colchicine or DMSO 42 h after release from DEX. Scale bar, 20 μm.

  6. Representative views of primary cilia for panel E. Scale bar, 2 μm

  7. Quantitative analysis of primary cilium length in NIH/3T3 cells treated with 200 nM colchicine or DMSO 30 or 42 h after release from DEX. The data are from four independent experiments.

  8. Representative images of PCM1 redistribution to cell periphery or center upon inducible dimerization PCM1 with the plus and minus end‐directed molecular motor proteins. Immunostaining for GFP in NIH/3T3 cells expressing GFP‐PCM1‐FKBP alone (indicated as Control), co‐expressing GFP‐PCM1‐FKBP with HA‐Kif5b‐FRB (indicated as Kif5b‐FRB) or HA‐BICD2‐FRB (indicated as BICD2‐FRB). Cells were treated with rapamycin for 1 h at 23 h after release from DEX and fixed 6 h later. Scale bar, 10 μm.

  9. Schematic diagram of the protocol for treatment of rapamycin (Rapa) to NIH/3T3 cells exposed to DEX.

  10. Immunostaining for GFP (green) and PCNT (red) in NIH/3T3 cells expressing indicated plasmid at 30 h after release from DEX. Scale bar, 5 μm.

  11. Quantitative analysis of primary cilium length in NIH/3T3 cells expressing indicated plasmids at 42 h after release from DEX. The data are from three independent experiments.

Data information: Data in panels C, G, and K are presented as mean ± SEM. **P ≤ 0.01, # P ≤ 0.05, and ## P ≤ 0.01 (one‐way ANOVA). Source data are available online for this figure.
Figure EV2
Figure EV2. Rhythmic dynamics of IFT88 in primary cilia
  1. A

    Immunostaining for primary cilia (Arl13b) and IFT88 in NIH/3T3 cells at 24 or 40 h after release from DEX. Scale bar, 2 μm.

  2. B, C

    Quantitative analysis of fluorescence intensity of IFT88 in primary cilia (Arl13b, B) and centrosome (γ‐tubulin, C) of NIH/3T3 cells at the indicated time point after release from DEX. The data are from three independent experiments.

Data information: Data in panels B and C are presented as mean ± SEM. **P ≤ 0.01 (one‐way ANOVA). Source data are available online for this figure.
Figure 6
Figure 6. Rhythmic dynamics of primary cilium length influence Hh signaling

  1. Schematic diagram of the protocol for treatment of SAG to NIH/3T3 cells exposed to DEX.

  2. Real‐time qPCR analysis of Gli1 or Ptch1 mRNA expression levels in the NIH/3T3 cells incubated with SAG for 4 h at 28 h or 40 h after release from DEX. The data are from three independent experiments.

Data information: Data in panel B are presented as mean ± SEM. *P ≤ 0.05 and **P ≤ 0.01; n.s. indicates no significant difference (one‐way ANOVA). Source data are available online for this figure.
Figure 7
Figure 7. Primary cilia influence fibroblast mobilization with circadian changes in wound healing
  1. A

    Schematic diagram of the protocol for wound healing assay using NIH/3T3 cells exposed to DEX.

  2. B

    Representative images of WT or Kif3a‐KO NIH/3T3 cells at 0 or 24 h after wounding in the wound healing assay with the protocol illustrated in panel A. Scale bar, 200 μm.

  3. C

    Quantitative analysis of the area of wound healing from panel B at the indicated time points after wounding. The data are from three independent experiments. Data are presented as the mean ± SEM. **P ≤ 0.01; n.s. indicates no significant difference (one‐way ANOVA).

  4. D

    Representative time‐lapse images of NIH/3T3 WT at the indicated time after wounding in the wound healing assay with the protocol illustrated in panel (A). Scale bar, 50 μm.

  5. E, F

    Quantitative analysis of the coordinates of the cellular centroid (E) and the moving distance of cells (F) from panel D.

Source data are available online for this figure.
Figure EV3
Figure EV3. Bbs2 knockout NIH/3T3 cells in wound healing

  1. Immunostaining for primary cilia (red; Arl13B) and DNA (blue; DAPI) in WT or Bbs2‐KO NIH/3T3 cells. Scale bar, 10 μm. White arrows point to primary cilia.

  2. Representative images of WT or Bbs2‐KO NIH/3T3 cells at 0 or 24 h after wounding in the wound healing assay with the protocol illustrated in Fig 7 panel A. Scale bar, 200 μm.

  3. Quantitative analysis of the area of wound healing from panel B at 24 h after wounding. The data are from three independent experiments.

Data information: Data in panel C are presented as mean ± SEM. *P ≤ 0.05; n.s. indicates no significant difference (one‐way ANOVA). Source data are available online for this figure.
Figure EV4
Figure EV4. Increased primary cilium length decreases cell migration

  1. Immunostaining for GFP in NIH/3T3 cells with forced expression of Arl13b‐venus (long cilia) or physiological expression of Arl13b‐venus (normal cilia) at 0 or 24 h after wounding in the wound healing assay. Scale bar, 5 μm.

  2. Representative images of NIH/3T3 cells with long cilia or normal cilia at 0 or 24 h after wounding in the wound healing assay. Scale bar, 200 μm.

  3. Quantitative analysis of the area of wound healing from panel B at 24 h after wounding. The data are from three independent experiments.

Data information: Data in panel C are presented as mean ± SEM. *P ≤ 0.05 (one‐way ANOVA). Source data are available online for this figure.
Figure 8
Figure 8. Rhythmic dynamics of primary cilia in wound healing

  1. Schematic diagram of experimental protocol after the wound healing assay for NIH/3T3 cells exposed to DEX.

  2. Representative fluorescence image of DAPI‐stained NIH/3T3 cells at 12 h after wounding using the protocol illustrated in panel (A). Scale bar, 200 μm.

  3. Immunostaining for primary cilia (red; Arl13b) in NIH/3T3 cells at the indicated area (I–III as in B) with the protocol illustrated in panel A. Scale bar, 20 and 2 μm in magnificent images.

  4. Quantitative analysis of primary cilium length from panel C. The data are from over 60 cells per condition from two individual experiments.

  5. Immunostaining for primary cilia (red; Arl13b), F‐actin (green; phalloidin), and DNA (blue; DAPI) in NIH/3T3 cells at 1 h after wounding with the protocol illustrated in panel A. Arrows indicate the direction and length between the nucleus and the migrating edge of cell. Scale bar, 20 μm.

  6. Quantitative analysis of the distance from the nucleus to the migrating edge of cell from panel E (white arrows in panel E). The data are from 27 cells per condition.

  7. Hypothetical schematic diagram of the relationship between primary cilia and cell migration showing circadian rhythms.

Source data are available online for this figure.
Figure EV5
Figure EV5. Primary cilia angle in wound healing

  1. Schematic diagram of the angle of primary cilia relative to the direction of the wound.

  2. Quantitative analysis of the angle of primary cilia from panel E in Fig 8. The data are from 19 cells at 18 h and 14 cells at 30 h.

Source data are available online for this figure.
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