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

受激发射耗尽(STED)显微镜是第一个打破衍射势垒并在大分子尺度上提供空间分辨率的显微镜概念。最近的实验已经证明了生物显微镜在20 nm处的次衍射分辨率(Donnert et.PNAS，2006)和绿色荧光(GFP-)标记细胞的STED显微镜(威廉等人。Al.，Nat.方法，2006)。使用多个荧光标记的双色STED显微镜将极大地有助于细胞中多蛋白质复合体的分析。初步实验和实际考虑表明，使用两种不同的荧光蛋白(XFP)进行双色STED是可行的。在这里提出的项目中，我们打算探索可能的最好的双色STED荧光蛋白质变体，并为生物学中纳米级(20 Nm)的基于XFP的多色显微镜铺平道路。特别是，我们将使用多色STED显微镜来研究哺乳动物细胞中的电压依赖阴离子通道(VDAC)。VDAC是线粒体介导的细胞凋亡途径中的一个关键角色。它是线粒体外膜的主要成分，以多蛋白复合体的形式组织。STED显微镜将被用来研究这些多蛋白复合体在健康细胞和凋亡细胞中的亚线粒体定位、动力学和组成。通过研究以前无法获得的空间分辨率的事件，我们期望在线粒体介导的细胞凋亡的事件链中获得新的见解。

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.