Untersuchung sub-mitochondrialer Proteinverteilungen in unbehandelten und apoptotischen humanen Zellen mittels STED-Mikroskopie

Abstract

Mitochondria are essential for eukaryotic cells. These organelles exhibit a complex internal structure and fulfil a variety of different tasks. They are known as the powerhouses of the cell because of their important role in the supply of the universal biological energy equivalent ATP. Although mitochondria belong to the best characterized organelles, only little is known about protein distributions within mitochondria. The light microscopic analysis of sub-mitochondrial protein distributions was neglected for a long time, because mitochondria have a diameter of 200-400 nm, which is in the range of the resolution limit of conventional light microscopes. However, recent developments of high resolution light microscopic techniques like STED-microscopy allow the investigation of sub-mitochondrial protein distributions. Up to now, only qualitative analysis of mitochondrial proteins and their distributions were performed. In this work STED-microscopy was combined with mathematical algorithms to obtain for the first time quantitative data on sub-mitocondrial protein distributions. By qualitative and quantitative analysis of fluorescence-microscopy based data, two different issues could be addressed: (1) The voltage-dependent anion-selective channel (VDAC) is a protein of the mitochondrial outer membrane mediating the transport of metabolites, including ATP. In humans, three different isoforms exist. Until now little is known about the differences between the three hVDAC isoforms. In this work it was shown that the three hVDAC isoforms have a different sub-mitochondrial localization. Further an amino acid residue was identified, which has an influence on the sub-mitochondrial localization of hVDAC. The results of quantitative colocalization analysis between the different hVDAC isoforms suggest the existence of VDAC-heterooligomers. Using quantitative STED-microscopy, it could be shown that the degree of colocalization between hVDAC and their possible interaction partner hexokinase-I is isoform specific. In addition, the data reveal that a substantial fraction of hexokinase-I does not colocalize with any of the hVDAC isoforms, suggesting a more complex interplay of these proteins than previously anticipated. (2) Apoptosis, one form of programmed cell death, is a fundamental process for the life of multicellular organisms. Despite this very important fact, little is known about how exactly cytochrome c is released from mitochondria, as well as the sub-mitochondrial protein distributions and possible changes during apoptosis. By using quantitative STED-microscopy, it could be shown that no massive changes in the protein distributions of six different mitochondrial proteins take place during early stages of apoptosis. Rather, subtle changes to more homogenous protein distributions were obeserved, which occur before the cytochrome c release. These results broaden the view on the apoptotic pathway: Not only subtle structural changes take place within mitochondria during apoptosis (as proven by other studies), but also subtle changes in the sub-mitochondrial protein distributions, which can be explained by the tendency of disintegration of protein clusters. With STED-microscopy it was hence possible to detect subtle changes in sub-mitochondrial protein distributions, which would otherwise remain undiscovered in conventional light microscopy.