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.2011 Feb 22;108(8):3175-80.
doi: 10.1073/pnas.1012842108. Epub 2011 Feb 7.

Nanopatterned protein microrings from a diatom that direct silica morphogenesis

Affiliations

Nanopatterned protein microrings from a diatom that direct silica morphogenesis

André Scheffel et al. Proc Natl Acad Sci U S A..

Abstract

Diatoms are eukaryotic microalgae that produce species-specifically structured cell walls made of SiO(2) (silica). Formation of the intricate silica structures of diatoms is regarded as a paradigm for biomolecule-controlled self-assembly of three-dimensional, nano- to microscale-patterned inorganic materials. Silica formation involves long-chain polyamines and phosphoproteins (silaffins and silacidins), which are readily soluble in water, and spontaneously form dynamic supramolecular assemblies that accelerate silica deposition and influence silica morphogenesis in vitro. However, synthesis of diatom-like silica structure in vitro has not yet been accomplished, indicating that additional components are required. Here we describe the discovery and intracellular location of six novel proteins (cingulins) that are integral components of a silica-forming organic matrix (microrings) in the diatom Thalassiosira pseudonana. The cingulin-containing microrings are specifically associated with girdle bands, which constitute a substantial part of diatom biosilica. Remarkably, the microrings exhibit protein-based nanopatterns that closely resemble characteristic features of the girdle band silica nanopatterns. Upon the addition of silicic acid the microrings become rapidly mineralized in vitro generating nanopatterned silica replicas of the microring structures. A silica-forming organic matrix with characteristic nanopatterns was also discovered in the diatom Coscinodiscus wailesii, which suggests that preassembled protein-based templates might be general components of the cellular machinery for silica morphogenesis in diatoms. These data provide fundamentally new insight into the molecular mechanisms of biological silica morphogenesis, and may lead to the development of self-assembled 3D mineral forming protein scaffolds with designed nanopatterns for a host of applications in nanotechnology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic primary structures of cingulins. The overall contents of Y or W in cingulins is substantially above the average frequency in proteins, which is 1.0% for W and 2.9% for Y (computed from proteins in UniProtKB/Swiss-Prot data bank). CinY1: 7.3% Y, 0% W; CinY2: 7.7% Y, 0% W; CinY3: 7.7% Y, 0% W; CinW1: 3.1% Y, 5.8% W; CinW2: 3.4% Y, 5.0% W; CinW3: 2.6% Y, 6.2% W.
Fig. 2.
Fig. 2.
Analysis of cellular localization, biosilica association, and solubility of cingulins and silaffin tpSil3. (A) (Top) Confocal fluorescence microscopy images of liveT. pseudonana transformant strains expressing GFP fusion proteins of cingulins and tpSil3. For each strain an individual cell is shown in transapical cross section. The red fluorescence is caused by chlorophyll. (Middle) Epifluorescence microscopy images of isolated biosilica from multiple cells. (Bottom) Epifluorescence microscopy images of ammonium fluoride insoluble material (AFIM) obtained from biosilica after complete demineralization. The numbers in the bottom right corner indicate the camera exposure times for recording the images. Scale bars: 5 μm. (B) Schematic of aT. pseudonana cell shown in transapical cross section. (C) Quantification of GFP fluorescence in AFIM relative to GFP fluorescence in the biosilica from which the AFIM was prepared.
Fig. 3.
Fig. 3.
SEM analysis of the AFIM and biosilica fromT. pseudonana. (A) Overview of the AFIM and (B) an individual microring from the same preparation. The dark dots that are present within and outside the microrings are the pores in the underlying filter membrane. (C) Intact biosilica from an individualT. pseudonana cell. The white dotted lines delineate the pore-free rims of the girdle bands. V: valve, Gb, girdle band.
Fig. 4.
Fig. 4.
Microring induced silica formation. (A) Time dependence of the amount of silica precipitated by constant amounts of microrings (chitin-free) in the absence (circles) and presence (triangles) of a constant concentration of the synthetic polyamine DAB-Am-16. Error bars indicate standard deviation. (B) Scanning electron micrographs of a microring before (Top) and a microring after (Bottom) incubation with silicic acid for 60 min in the presence of synthetic polyamine. Both images were recorded with the secondary electron detector (SED). The SED is more sensitive to surface morphology than the inlense detector (ILD) enhancing the contrast of the contour and the appearance of surface roughness in the recorded images (Note: The ILD was used for recording the scanning electron microscopy images in Fig. 3). In theUpper panel (before remineralization) the fibers of the microring appear as dark flat strips that run roughly parallel in vertical direction. In theLower panel (after remineralization) gray strips are visible, which are raised above the background, and run roughly parallel in vertical direction. The dark dots that are present within and outside the microrings are caused by the pores in the underlying filter membrane. (C) Atomic force microscopy images of a microring before (Top) and a microring after (Bottom) incubation with silicic acid for 60 min in the presence of synthetic polyamine. The images in the middle column are higher magnifications of the images in the left column. The right column shows the height profiles along the numbered lines that are indicated in the images of the middle column. The lines were located on the background (Si wafer, line 1), the organic material between two fibers (line 2), and along an individual fiber (line 3). The numbers on the right side of the height profile diagrams indicate the average heights and standard deviations in nm of the profiles along the numbered lines.
Fig. 5.
Fig. 5.
Model for girdle band biogenesis inT. pseudonana. The schematics depict details from a cell in transapical cross section. SDV = silica deposition vesicle. Silica structures are shown in green. (Step 1): An insoluble cingulin-containing nanoring is present in a developing girdle band SDV. The shaping of the SDV membrane and positioning of the girdle band SDV is presumably under control of the cytoskeleton (23). (Step 2): Following uptake of silicic acid into the SDV the nanoring together with soluble organic components (e.g., LCPA, silaffins, silacidins) templates formation of the girdle band silica inside the SDV. (Step 3): After exocytosis the newly formed girdle band is attached to the girdle band region by enzyme catalyzed fusion with the cingulin-containing nanoring of the neighboring girdle band.
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