The AC3362 protein resembles the cingulins from the diatom T. pseudonana, which are also rich in tyrosine residues. As the AC3362-YFP fusion protein was accessible to antibody molecules in immunolabeling it appears to be partially or fully exposed on the Bortezomib biosilica surface, rather than completely embedded within the biosilica. Ammonium fluoride-insoluble organic matrices that are composed of proteins and polysaccharides and exposed on the biosilica surface have recently been identified in several diatoms. Tesson and Hildebrand argued that insoluble organic matrices embedded within diatom biosilica may not exist as they were unable to detect ammonium fluoride-insoluble organic matrices from acid hydrolyzed biosilica. However, their data were inconclusive, because their argument was based on the resistance to acid hydrolysis of the biosilica-associated long-chain polyamines, whichare devoid of acid-labile bonds. Therefore, it cannot be argued that silica-embedded organic matrices have to be resistant to acid hydrolysis conditions. In contrast, as biosilica is highly porous and hydrated it can be expected that protons can easily penetrate throughout the biosilica, and thus polysaccharideand protein-based insoluble organic-matrices should become completely degraded under acid hydrolysis conditions, regardless of whether they are embedded within or exposed on the surface of biosilica. In future research, analysis by immunofluorescence microscopy, using the method described in the present study, may be able to validate whether the cingulin-containing organic matrix of T. pseudonana is embedded inside the biosilica or located on the biosilica surface. It has been discussed that biosilica-associated organic matrices may have a role in biosilica formation, mechanical support of biosilica, the stabilization of biosilica against dissolution, or combinations of these functions. One possibility to further investigate the function of the AC3362 containing organic matrix would be the generation of knock-down mutants. This technique is established for the diatoms P. tricornutum and T. pseudonana, and should be also possible for A. coffeaeformis by utilizing the transformation system that has been established in this study. The bioinformatics screen for mussel-like putative adhesion proteins in A. coffeaeformis has yielded three additional tyrosinerich proteins. However, we were unable to detect by fluorescence microscopy the production of the resulting YFP fusion proteins in A. coffeaeformis transformants. Expression rate of the YFP fusion proteins may have been too low, the C-terminal YFP-tag may be proteolytically cleaved from the mature protein, or the YFP domain may have interfered with folding or stability of these tyrosine-rich proteins. Whatever the case, due to the presence of the encoding mRNAs it is reasonable to assume that the three proteins are produced in A. coffeaeformis wild type cells.