Figure 1: Molecular mechanisms of silica scale production in P. neolepis.

(a) Differential interference contrast (DIC) microscopy image of P. neolepis cells displaying the loose covering of silica scales. Scale bar, 10 μm. (b) Confocal microscopy of a P. neolepis cell showing incorporation of the fluorescent dye HCK-123 into newly formed silica scales (green). Chlorophyll autofluorescence is shown in red. The 3D-projection was generated from compiling a Z-stack of 15 images. Scale bar, 10 μm. (c) Tricine/SDS–PAGE of organic components released after dissolution of silica scales with NH₄F. A SEM image of an isolated silica scale is also shown (Scale bar, 1 μm). The higher molecular weight component around 50 kDa is a single protein that runs as two bands (i, ii), whereas the low-molecular-weight components around 2.5 kDa are long-chain polyamines (LCPA). M, molecular-weight markers. (d) Domain organization of the lipocalin-like protein (LPCL1) identified from both protein bands in NH₄F extracted silica scales. The approximate positions of the proline/lysine-rich regions and the calycin domain (IPR012674) are shown. Also shown are the positions of six highly conserved cysteines (asterisk) that may be involved in the formation of disulphide bridges. (e) Long-chain polyamines (LCPAs) from P. neolepis silica scales. Electrospray ionization mass spectrometry (ESI-MS) of the low-molecular-weight NH₄F-soluble fraction of silica scales revealed a series of mass peaks separated by 71 Da (highlighted in red), characteristic of N-methyl propyleneimine units. The additional mass peaks ±14 Da may indicate different methylation states, as is commonly observed in LCPAs. The proposed structure of the LCPAs in P. neolepis is shown with the putative lysine residue is highlighted in red.