Analysis of VDAC multi-protein complexes with dual-color fluorescent protein nanoscopy

双色荧光蛋白纳米镜分析 VDAC 多蛋白复合物

• 批准号: 5450169
• 负责人: Professor Dr. Stefan Jakobs
• 金额: --
• 依托单位: Klinik für Neurologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2005
• 资助国家: 德国
• 起止时间: 2004-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5450169?language=en
• 关键词: Analysis; VDAC; multi; protein; complexes

Stimulated emission depletion (STED-) microscopy is the first microscopy concept to break the diffraction barrier and to provide spatial resolution at the macromolecular-scale. Recent experiments have demonstrated subdiffraction resolution at < 20 nm in biological microscopy (Donnert et. al., PNAS, 2006) and STED microscopy with green fluorescent (GFP-) labeled cells (Willig et. al., Nat. Methods, 2006). The analysis of multi-protein complexes in cells will greatly benefit from dual-color STED microscopy employing multiple fluorescence markers. Initial experiments and practical considerations suggest the feasibility of dual-color STED using two different fluorescent proteins (XFPs). In the proposed project here, we intend to explore the best possible dual-color STED variants of fluorescent proteins and to pave the way for multicolor XFP-based microscopy at the nanoscale (< 20 nm) in biology. In particular, we shall employ multicolor STED microscopy to investigate the Voltage-Dependent Anion Channel (VDAC) in mammalian cells. VDAC is a key player in the mitochondria-mediated apoptosis pathway. It is a major constituent of the outer mitochondrial membrane and is organized in multiprotein complexes. STED microscopy will be utilized to investigate the sub-mitochondrial localizations, dynamics and compositions of these multi-protein complexes in healthy and apoptotic cells. By investigating events with a previously not accessible spatial resolution, we expect to obtain new insights in the chain of events in mitochondria-mediated apoptosis.

受激发射耗尽（STED）显微镜是第一个显微镜概念，打破衍射障碍，并提供在大分子尺度的空间分辨率。最近的实验已经证明了在生物显微镜中< 20 nm的亚衍射分辨率（Donnert et.例如，PNAS，2006）和使用绿色荧光（GFP-）标记的细胞的STED显微术（Willig et.例如，方法，2006）。细胞中多蛋白复合物的分析将大大受益于采用多个荧光标记的双色STED显微镜。初步实验和实际考虑表明，双色STED使用两种不同的荧光蛋白（XFP）的可行性。在这里提出的项目中，我们打算探索荧光蛋白的最佳双色STED变体，并为生物学中纳米级（< 20 nm）的基于XFP的显微镜铺平道路。特别是，我们将采用荧光显微镜来研究哺乳动物细胞中的电压依赖性阴离子通道（VDAC）。VDAC是细胞凋亡途径中的关键参与者。它是线粒体外膜的主要组成部分，并以多蛋白复合物的形式组织。STED显微镜将用于研究健康和凋亡细胞中这些多蛋白复合物的亚线粒体定位，动力学和组成。通过调查以前无法访问的空间分辨率的事件，我们希望获得新的见解，在链的事件中的细胞凋亡。