SP-5: Single-Molecule Analysis of Biomolecule Assemblies

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Biomineralization in diatoms is a highly complex process to create the intricate and extremely regular and reproducible nanoscale pattern of silica in diatom cell walls. We hypothesize that biosilica morphogenesis depends on the patterning (in time and space) of proteins (silaffins, silacidins, cingulins, silicanins) in combination with long-chain polyamines (LCPA). Localizing these proteins as exactly as possible and correlating these to the silica patterns is thus crucial to verify this hypothesis and gain further fundamental understanding of this patterning process. In the past funding period, we have developed single-molecule localization microscopy for the diatom T. pseudonana based on photo-activatable localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). We have identified a number of different photo-convertible fluorescent proteins, which allow as fusion proteins to localize silica embedded proteins with a precision down to 25 nm (approximately a 10-fold improvement compared to confocal microscopy). We have further applied SMLM to study the insoluble organic matrix cingulin proteins. Here, while epifluorescence images suggest a rather continuous distribution of both proteins, the reconstructed super-resolution images showed regions of CinW2 and CinY2 fluorescence patches. In this project, we aim to address the following questions using single-molecule fluorescence detection: 1) What patterns are formed by the silicanin transmembrane proteins with respect to the cingulin proteins in microrings? 2) What is the pattern of the soluble components on the insoluble organic matrix? 3) What is the pattern of Silicanin-1 in the SDV and during biosilica formation? The first project will focus on in vitro systems using PALM and STORM microscopy to localize Silicanin-1. We will further develop correlative light and electron microscopy (CLEM) to position the reconstructured super-resolution data on high-resolution, yet protein unspecific, electron micrographs. The second package will localize with super-resolution microscopy in a protein unspecific manner the self-assembly of soluble organic components on the insoluble organic matrix. In a third work package, we will establish imaging conditions for in vivo super-resolution to visualize pattern formation of the transmembrane protein silicanin-1 in the silica deposition vesicle during the valve biogenesis in the cleavage furrow.