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

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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.