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Silicanin-1 is a conserved diatom membrane protein involved in silica biomineralization - PubMed

  • ️Sun Jan 01 2017

Silicanin-1 is a conserved diatom membrane protein involved in silica biomineralization

Alexander Kotzsch et al. BMC Biol. 2017.

Abstract

Background: Biological mineral formation (biomineralization) proceeds in specialized compartments often bounded by a lipid bilayer membrane. Currently, the role of membranes in biomineralization is hardly understood.

Results: Investigating biomineralization of SiO2 (silica) in diatoms we identified Silicanin-1 (Sin1) as a conserved diatom membrane protein present in silica deposition vesicles (SDVs) of Thalassiosira pseudonana. Fluorescence microscopy of GFP-tagged Sin1 enabled, for the first time, to follow the intracellular locations of a biomineralization protein during silica biogenesis in vivo. The analysis revealed incorporation of the N-terminal domain of Sin1 into the biosilica via association with the organic matrix inside the SDVs. In vitro experiments showed that the recombinant N-terminal domain of Sin1 undergoes pH-triggered assembly into large clusters, and promotes silica formation by synergistic interaction with long-chain polyamines.

Conclusions: Sin1 is the first identified SDV transmembrane protein, and is highly conserved throughout the diatom realm, which suggests a fundamental role in the biomineralization of diatom silica. Through interaction with long-chain polyamines, Sin1 could serve as a molecular link by which the SDV membrane exerts control on the assembly of biosilica-forming organic matrices in the SDV lumen.

Keywords: Biomineralization vesicles; Cryo-TEM; Diatom biosilica; Exocytosis; Protein self-assembly; Silica formation activity; Time-lapse confocal fluorescence microscopy; Transmembrane protein; Vesicle biogenesis.

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Figures

Fig. 1
Fig. 1

Sequence analysis of Sin1. a Analysis of the amino acid sequence of Sin1. The signal peptide is depicted in italics and underlined, the RXL domain is highlighted in purple, and clusters that are rich in N and Q are presented on a red background. The transmembrane helix is highlighted in orange and the cytosolic domain in blue. The N-terminal signal peptide and the transmembrane helix were identified using the SignalP v.4.1 [47] and TMHMM v.2 [48] webservers, respectively. b Schematic of the domain arrangement in Sin1. SP signal peptide, RXL RXL domain, tm transmembrane helix, cyt cytosolic domain

Fig. 2
Fig. 2

Western blot analysis using anti-Sin1 antibodies. M total membrane fraction from T. pseudonana; M + HF total membrane fraction from T. pseudonana after treatment with anhydrous HF; a Apparent molecular mass of native Sin1 in comparison to recombinant proteins rSin1-SP and rSin1lum (Additional file 1: Figure S10). b Effect of anhydrous HF on the apparent molecular mass of native Sin1. The left lanes in a and b contain molecular mass standard proteins

Fig. 3
Fig. 3

Localization of Sin1-GFP fusion proteins in T. pseudonana. a SEM images of biosilica from individual cells in two different orientations. b Live cells, biosilica, and biosilica-associated organic matrix from transformant strains expressing Sin1-GFPN or Sin1-GFPC. The fusion proteins were expressed under control of the endogenous Sin1 promoter and terminator sequences. The ‘Live cell’ panels show confocal fluorescence images (z-projection) of individual cells in girdle view (left panel, and third panel from the left) and in valve view (second panel from the left). Green color indicates the GFP fusion proteins and the red color is caused by chlorophyll autofluorescence. The biosilica and organic matrix panels show bright field microscopy images (BF) and the corresponding epifluorescence microscopy images (EF) of material isolated from Sin1-GFPN- or Sin1-GFPC-expressing transformants. Scale bars for all images: 2 μm. c Proposed intracellular proteolytic processing of Sin1. Sin1 becomes cleaved by a protease between the luminal domain (lum) and the transmembrane helix (orange). The luminal domain is incorporated into the biosilica, while the transmembrane helix and the cytosolic domain (blue squiggle) become degraded

Fig. 4
Fig. 4

Localization of Sin1-GFPC around the time of valve biogenesis. a Schematic drawings illustrating the different stages of the diatom cell cycle. For simplicity, intracellular compartments, except for the SDVs, have been omitted. Black and blue colors indicate mature biosilica and newly produced biosilica, respectively. Red and yellow colors depict the plasma and SDV membranes, respectively. b–d Selected images from time-lapse confocal fluorescence microscopy of Sin1-GFPC labeled with PDMPO are shown (Additional file 2: Movie S1). The time above the images relates to the peak of the GFP and PDMPO fluorescence (Fig. 5), which is set as t = 0 min. Panel b shows the GFP fluorescence (green), panel c the PDMPO fluorescence (blue), and panel d an overlay of GFP and PDMPO fluorescence (note: a superposition of green and blue fluorescence appears cyan). All images are projections of nine z-planes. Scale bars: 2 μm

Fig. 5
Fig. 5

Time-dependent quantitative analysis of region-specific (a) GFP and (b) PDMPO fluorescence during valve formation in Sin1-GFPC expressing cells. The plots show the averaged results from four different transformant cells labeled with PDMPO. From each cell, images were recorded in 3.5 min intervals, and the fluorescence intensities in different regions of the cell were determined. From each frame, z-projections were generated by combining all nine z-planes. The schematic shows the delineations of the cellular regions analyzed. The coloring of the cellular regions in the schematic corresponds to the line coloring in the graphs. The black lines represent the sum of the intensities from all regions of the cell. The frame with the maximum GFP and PDMPO fluorescence during the time-lapse recording was defined as t = 0 min. This allowed alignment of the time scale of the four different cells used in our analysis. The gray-shaded areas represent the standard deviation of the averaged fluorescence intensities obtained from the four cells for each region. No standard deviation is given for time periods for which only a single cell was available for averaging

Fig. 6
Fig. 6

Dynamic light scattering analysis of rSin1lum at different pH. A solution of rSin1lum was adjusted to increasingly acidic pH values (black traces), and then titrated back to near neutral pH (blue trace). The black traces show the particle distribution by mass. The dotted lines show the particle distribution by intensity to highlight the presence of small amounts of rSin1lum clusters

Fig. 7
Fig. 7

Cryo-electron microscopy analysis of rSin1lum clusters. a Clusters at different pH values 60 min after adjustment to the indicated pH. The black arrows point to small clusters that consist of only 10–20 protein monomers. b Clusters at pH 5.2 (left) and pH 5.0 (right). The arrowheads point to neck regions between two clusters that are indicative of fusion events

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