CRISPR-Cas9-mediated protein tagging in human cells for RESOLFT nanoscopy and the analysis of mitochondrial prohibitins
von Michael Ratz
Datum der mündl. Prüfung:2015-12-17
Betreuer:Prof. Dr. Stefan Jakobs
Gutachter:Prof. Dr. Stefan Jakobs
Gutachter:Prof. Dr. Peter Rehling
EnglischEctopic overexpression of fluorescent fusion proteins for live cell imaging studies often leads to a multitude of artefacts, but protein expression at endogenous levels in mammalian cells was difficult to achieve so far. To avoid common problems associated with overexpression, this study used the CRISPR-Cas9 genome engineering system for site-specific endogenous protein tagging in human cells. First, a general workflow for genome editing was established and then applied to generate heterozygous and homozygous human knock-in cells that express a fluorescent fusion from a genomic locus. Three human genes (HMGA1, VIM and ZYX) were tagged with the reversibly switchable fluorescent protein rsEGFP2 and the benefit of endogenous over ectopic expression demonstrated using flow cytometry and confocal microscopy. Moreover, low light intensity RESOLFT super-resolution microscopy could be applied to study nanoscale protein dynamics at physiologically relevant protein expression levels in living knock-in cells. CRISPR-mediated endogenous tagging was crucial to investigate the localization, dynamics and abundance of mitochondrial prohibitin 1 and 2, PHB1 and PHB2, in human cells. While overexpression of PHB1 and PHB2 caused aberrant mitochondria, endogenous tagging of prohibitins with the fluorescent protein Dreiklang (DK) restored wildtype mitochondrial morphology. Overexpression of PHB2-DK and human estrogen receptor α caused a mislocalization of PHB2-DK in the nucleus of HeLa cells, but also this artefact was not observed in endogenously tagged PHB2-DK HeLa cells. Homologous recombination frequencies for PHB1 and PHB2 tagging were remarkably high and a number of heterozygous PHB1-DK and PHB2-DK knock-in clones could be generated. STED super-resolution microscopy uncovered the hitherto unknown organization of individual prohibitins into clusters. Dual-color STED imaging demonstrated a colocalization of tagged and untagged PHB1 and PHB2 indicating that PHB complex formation is not affected by protein tagging. Intriguingly, the vast majority of prohibitins is found at the mitochondrial cristae membrane where they form exceptionally static protein assemblies. Surprisingly, the global amount of PHB2 was found to be 4-5 times higher than that of PHB1, which is in contrast to in vitro studies conducted on purified yeast prohibitins. After integrating the results on prohibitin localization and abundance with morphological data about the ultrastructural organization of mitochondria and under the assumption that human prohibitins form a 1 MDa complex, it was estimated that about 31-36 individual PHB complexes occupy a single cristae membrane.
Keywords: CRISPR, RESOLFT, prohibitin, mitochondria, STED, gene editing, nanoscopy, super-resolution