SP-5: Single-Molecule Analysis of Biomolecule Assemblies

硅藻中的生物矿化是一个高度复杂的过程，要在硅藻细胞壁中产生错综复杂、极其规则和可重复的纳米级二氧化硅图案。我们假设生物硅的形态发生依赖于蛋白质(silaffins，Silacidins，Cingulins，Silicanins)与长链多胺(LCPA)的组合(在时间和空间上)的模式。因此，尽可能准确地定位这些蛋白质并将其与二氧化硅图案相关联，对于验证这一假设并进一步从根本上理解这种图案化过程至关重要。在过去的资助期间，我们在光激活定位显微镜(Palm)和随机光学重建显微镜(STORM)的基础上发展了硅藻假单藻的单分子定位显微镜。我们已经确定了许多不同的光可转换荧光蛋白，这些融合蛋白使AS能够定位硅胶嵌入蛋白，定位精度低至25 nm(与共聚焦显微镜相比，大约提高了10倍)。我们进一步将SMLM应用于不溶性有机基质扣带蛋白的研究。在这里，尽管荧光图像显示这两种蛋白质的分布相当连续，但重建的超分辨率图像显示了CinW2和CinY2荧光斑块的区域。在这个项目中，我们的目标是利用单分子荧光检测来解决以下问题：1)硅胶跨膜蛋白相对于微粉中的刺参蛋白形成了什么图案？2)不溶有机基质上的可溶性成分是什么图案？3)在SDV和生物硅胶形成过程中，硅胶-1的图案是什么？第一个项目将专注于使用Palm和Storm显微镜对水飞蓟素-1进行定位的体外系统。我们将进一步发展相关光学和电子显微镜(CLEM)，将重建的超分辨率数据定位在高分辨率、但蛋白质非特异性的电子显微镜上。第二个包将用超分辨率显微镜以蛋白质非特异性的方式定位，即可溶性有机成分在不可溶有机基质上的自组装。在第三个工作包中，我们将建立体内超分辨率的成像条件，以可视化在卵裂沟中瓣膜生物发生过程中硅沉积囊泡中跨膜蛋白Silicanin-1的图案形成。

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.