Research ArticleBIOMATERIALS

Control of nacre biomineralization by Pif80 in pearl oyster

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Science Advances  02 Aug 2017:
Vol. 3, no. 8, e1700765
DOI: 10.1126/sciadv.1700765
  • Fig. 1 Bacterial production and Ca2+-induced coacervation of rPif80.

    (A to C) Analyses of expressed rPif80 in E. coli by SDS-PAGE with Coomassie staining (A), Western blot detection of the His6 tag in cell lysate (B), and purified rPif80 by SDS-PAGE with Coomassie staining (C). Lanes: M, protein molecular weight marker; S, soluble fraction; I, insoluble fraction; P, purified rPif80. (D) MALDI-TOF MS analysis of purified rPif80. a.u., arbitrary units. (E and F) Optical micrograph of the coacervate droplets of rPif80 in solutions of CaCl2 (E) and supersaturated CaCO3 (F). (G) Turbidimetric measurements of rPif80 coacervation according to Ca2+ concentration in solutions of CaCl2 and supersaturated CaCO3. Turbidity of the supersaturated CaCO3 solution in the absence of rPif80 was measured to exclude the possible influence of mineral growth on the increase of turbidity. All of the measurements were performed in triplicate. CaCl2-rPif80, coacervation of rPif80 in CaCl2 solution; sCaCO3-rPif80, coacervation of rPif80 in supersaturated CaCO3 solution; sCaCO3, supersaturated CaCO3 solution.

  • Fig. 2 rPif80-induced CLP.

    (A) Optical micrograph of rPif80-CLP droplets in the presence of Ca2+ and CO32−/HCO3. (B) Cryo-TEM image (left) and illustration (right) of rPif80-CLP droplets in (A). Dense granules of PILP-like ACGs are shown inside rPif80-CLP with cloudy morphology. (C) Cryo-scanning TEM image and electron diffraction pattern (inset) of rPif80-CLP droplets after initial formation (left) and a 4-day incubation at 4°C (right). (D) Raman spectra of the rPif80-CLP after initial formation and a 4-day incubation at 4°C. The Raman spectrum of an Si wafer is presented for comparison. The red arrows indicate the ν1 vibration mode of CaCO3.

  • Fig. 3 Morphology and polymorph analyses of grown minerals obtained by in vitro CaCO3 crystallization on β-chitin.

    (A to F) SEM images of grown minerals after crystallization at 20°C for 48 hours in the presence of rPif80 at concentrations of 0 (A), 15 (B), 30 (C), 50 (D), 100 (E), and 150 μg/ml (F). The insets of (A), (D), and (E) are magnified images of the selected areas indicated by the red arrowheads. (G) Raman spectra of grown minerals in (A) to (F) (left), and enlarged spectra from the red box (right). The Raman spectrum of calcite powder is presented for comparison. The red dashed lines correspond to the Raman peaks of aragonite, and the blue dashed line indicates the shift in the ν1 vibration mode of CaCO3.

  • Fig. 4 Schematic illustration of the proposed roles of Pif80 in the nacre formation in P. fucata.

    Nacre formation is processed through the following steps: (i) PILP-like ACGs are formed in intracellular vesicles and stably stored as a form of Pif80-CLP by a Ca2+-Pif80 coacervate. (ii) The PILP-like ACGs are destabilized by the disruption of the Ca2+-Pif80 coacervate in the extrapallial space and supplied for crystallization of nacre. (iii) Nacreous aragonites are grown on the β-chitin substrate using redissolved Pif80, eventually leading to the maturation of polygonal tablets.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1700765/DC1

    fig. S1. PTM analyses of native Pif80.

    fig. S2. Turbidimetric measurement of Ca2+-induced coacervation of rPif80 in the presence of 4 mM CaCl2.

    fig. S3. SDS-PAGE analysis with Stains-All staining of rPif80.

    fig. S4. Turbidimetric measurement of Ca2+-rPif80 coacervates according to additional NaCl.

    fig. S5. Optical micrograph images (top) and Raman spectra (bottom) of mineralized CaCO3.

    fig. S6. Cryo-scanning TEM image and EDS mapping analyses of rPif80-CLP.

    fig. S7. Dot blotting with Coomassie staining after CaCO3-binding analysis of rPif80.

    fig. S8. Structural analyses of a cross-sectioned plate mineral induced by rPif80 at a concentration of 50 μg/ml.

    fig. S9. Morphology and polymorph analyses of grown minerals in the presence of protein impurities.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. PTM analyses of native Pif80.
    • fig. S2. Turbidimetric measurement of Ca2+-induced coacervation of rPif80 in the presence of 4 mM CaCl2.
    • fig. S3. SDS-PAGE analysis with Stains-All staining of rPif80.
    • fig. S4. Turbidimetric measurement of Ca2+-rPif80 coacervates according to additional NaCl.
    • fig. S5. Optical micrograph images (top) and Raman spectra (bottom) of mineralized CaCO3.
    • fig. S6. Cryo-scanning TEM image and EDS mapping analyses of rPif80-CLP.
    • fig. S7. Dot blotting with Coomassie staining after CaCO3-binding analysis of rPif80.
    • fig. S8. Structural analyses of a cross-sectioned plate mineral induced by rPif80 at a concentration of 50 μg/ml.
    • fig. S9. Morphology and polymorph analyses of grown minerals in the presence of protein impurities.

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