Untersuchung des peroxisomalen Protein-Importes mittels super hochauflösender Lichtmikroskopie in der Hefe Saccharomyces cerevisiae
Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Zusammenfassung der Projektergebnisse
Peroxisomes are single membrane bound organelles with multiple cellular functions like βoxidation of fatty acids, the detoxification of reactive oxygen species and they are involved in many important biosynthetic pathways, like the biosynthesis of plasmologens. The multiple functions of peroxisomes depend on organism, cell type and function and can be adapted rapidly to changes in the cell environment. To fulfil all these different functions peroxisomes need to import their proteins posttranslational. Peroxisomes are able to import folded and even oligomerized proteins, which distinguishes this import machinery from import in other cell organelles. Most peroxisomel matrix proteins carry a peroxisomal targeting signal type1 (PTS1), which gets recognized in the cytosol by the peroxisomal import receptor PEX5. The receptor-cargo complex gets directed to the peroxisomal membrane, where PEX5 interact with its docking partner PEX14 and gets integrated into the peroxisomal membrane. Here PEX5 and PEX14 build a transient translocation pore through which the PTS1 cargo proteins are translocated in a by now unknown manner. In my work I am studying the import of peroxisomal proteins into the peroxisomal matrix in (living) cells using advanced optical microscopy tools. Specifically, I was interested in investigating protein distributions at the peroxisomal membrane and the dynamics of the PEX5-cargo interactions. One aim was to establish yeast as a model organism for investigation with super resolution STED microscopy. Yeast is a well-established model organism for investigation of peroxisomal biogenesis and protein import and a lot of well characterized genetic modifications are available. After a lot of optimization I was able to obtain STED images of fixed yeast cells with antibody labelling. Unfortunately, this approach requires the fixation of cells and is therefore not suitable for imaging of live cell dynamics. Therefore I tried to use photo stable dyes, which were transported across the cell wall of the yeast cells by electroporation. Unfortunately, again I was not able to obtain accurate-enough labelling despite of many optimization experiments. Due to the labelling problems with yeast I finally decided to introduce the desired genetic modifications into human cell lines using CRISPR/Cas9. Therefore, I created deletion cell lines of factors involved in the import of peroxisomal matrix protein import. In human cells we already established a labelling procedure and an image analysis pipeline for investigation of the distribution of membrane proteins at peroxisomes. Here, we found that in some peroxisomes the components of the translocation pore PEX5 and PEX14 seem to be separated (compartmentalized) at the peroxisomal membrane, while in most of the cases they show a high co-localization. Until now we were not able to link these different states to a physiological function but my newly created deletion cell lines will give us the opportunity to study for example modifications in the involved proteins. Besides such imaging studies in fixed cells, we also established the measurement of dynamics in living cells. Here, we used fluorescence (cross) correlation spectroscopy (FC(C)S) to measure the dynamics of PEX5-PTS1 interactions in the cytosol and I am investigating the movement of peroxisomes and their interaction with the cytoskeleton. Here, I established a method to track movement of peroxisomes with a high image frame-rate and an optimized tracking algorithm. At this point, I was able to distinguish precisely between short-distance vibrational movements and long-distance, tubulin-mediated directed movement of peroxisomes. In combination with the new deletion cell lines we are now able to link this different types of movement to the functionality of the peroxisomes. Taken together, I helped to establish different advanced-microscopy based approaches to study the dynamics of peroxisomal protein import. Moreover, I created cell lines that will allow us to us to modify this processes genetically, giving a strong outline for publication in the near future.
Projektbezogene Publikationen (Auswahl)
- (2016) Super resolution microscopy reveals compartmentalization of peroxisomal membrane proteins. J Biol Cem: M116.734038
Galiani S, Waithe D, Reglinski K, Cruz-Zaragoza LD, Garcia E, Clausen MP, Schliebs W, Erdmann R, Eggeling C
(Siehe online unter https://dx.doi.org/10.1074/jbc.M116.734083)
