Silica Nano- to Micro-Patterning at the Membrane Interface in vitro

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Silica biomineralization in diatoms takes place in highly specified organelles, the silica deposition vesicles (SDVs). A number of soluble (silaffins, silacidins, long chain polyamines) as well as insoluble bioorganic molecules (chitin, cingulins) have been found to be associated with biosilica, which is formed in the SDV and then exocytosed. However, as no protocol has been developed so far to isolate SDVs from any silica-forming organism, the processes occurring within the SDV as well as the composition of the surrounding lipid membrane and its influence on biological silica morphogenesis remain elusive. It is the aim of this project to understand the influence of lipid bilayers mimicking SDV membranes on biomolecule induced silica morphogenesis. In the previous funding period, we have successfully established supported membranes composed of complex lipid mixtures including diatom specific lipids. With these membranes in hand, we were able to analyze the interaction of cingulins, i.e. cingulin W2 and cingulin Y3, and of long-chain polyamines (LCPA). While cingulins do not bind to these membranes, LCPA interact with lipid bilayers resulting in the formation of three-dimensional membrane stacks. By a systematic study using different LCPA synthesized in the Geyer group (SP-6), we were able to show that a minimum number of amine groups (N > 5) is required to induce membrane stack formation. In the upcoming funding period, we will focus on silaffins and the SDV membrane associated protein silicanin-1 (Sin1), newly identified by members of this Research Unit 2038 in the previous funding period. In the first part of this proposal, we plan to recombinantly express, isolate and reconstitute full length Sin1 from Thallasiosira pseudonana in membranes. We will investigate the organization and self-aggregation properties of membrane-confined Sin1 as a function of pH making use of the membrane systems that we have developed in the previous funding period. Moreover, the silica precipitation properties of membrane confined Sin1 in the absence and presence of LCPA will be examined. To obtain information about the species specificity of the Sin1 protein, we plan to compare the membrane specific properties of Sin1 from T. pseudonana with those of Cylindrotheca fusiformis. In the second part of this proposal, we will analyze synthetic silaffin 1 derivatives provided by the Geyer group (SP-6) with different charge patterns with respect to their membrane binding ability and silica precipitation properties. The results will be compared with those obtained with native silaffin 1A1.