SP-5: Single-Molecule Analysis of Biomolecule Assemblies

SP-5：生物分子组装体的单分子分析

• 批准号: 249432622
• 负责人: Professor Dr. Michael Schlierf
• 金额: --
• 依托单位: B CUBE - Center for Molecular Bioengineering
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/249432622?language=en
• 关键词: SP; Single; Molecule; Analysis; Biomolecule

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.

硅藻的生物矿化是一个高度复杂的过程，它在硅藻细胞壁中产生了复杂的、极其规则的、可重复的纳米级二氧化硅图案。我们假设生物二氧化硅的形态发生取决于蛋白质（硅蛋白、硅酸蛋白、环藻蛋白、硅蛋白）与长链多胺（LCPA）结合的模式（在时间和空间上）。因此，尽可能精确地定位这些蛋白质并将它们与二氧化硅模式相关联，对于验证这一假设和进一步了解这一模式过程至关重要。在过去的资助期内，我们在光活化定位显微镜（PALM）和随机光学重建显微镜（STORM）的基础上开发了假硅藻单分子定位显微镜。我们已经确定了许多不同的光转换荧光蛋白，它们允许融合蛋白定位二氧化硅嵌入蛋白，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环蛋白的研究。在这里，虽然表观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荧光斑块区域。在这个项目中，我们的目标是利用单分子荧光检测解决以下问题：1)硅蛋白跨膜蛋白相对于微环中的环蛋白形成了什么模式？2)不溶性有机基质上可溶组分的分布规律是什么？3)硅酸-1在SDV和生物二氧化硅形成过程中的分布规律是什么？第一个项目将侧重于使用PALM和STORM显微镜在体外系统中定位硅蛋白-1。我们将进一步发展相关的光学和电子显微镜（CLEM），将重建的超分辨率数据定位到高分辨率，但蛋白质非特异性的电子显微镜上。第二个包将定位与超分辨率显微镜在蛋白质非特异性的方式，可溶性有机成分的自组装在不溶性有机基质。在第三个工作包中，我们将建立体内超分辨率成像条件，以可视化解理沟中阀门生物发生过程中二氧化硅沉积囊泡中跨膜蛋白硅蛋白-1的模式形成。