Investigation of the Mitochondria-ER axis and its interconnection with the MICOS complex

by Anna Laura Wittek
Doctoral thesis
Date of Examination:2024-09-10
Date of issue:2024-09-19
Advisor:Prof. Dr. Stefan Jakobs
Referee:Prof. Dr. Stefan Jakobs
Referee:Prof. Dr. Ralph Kehlenbach
Persistent Address: http://dx.doi.org/10.53846/goediss-10746

 

 

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Abstract

English

Mitochondria-ER contact sites (MERCS) are essential cellular hubs for the exchange of signaling molecules, lipids and metabolites. A multitude of MERCS-associated proteins have been described in mammalian cells, and rising evidence links MERCS with the inner mitochondrial membrane-shaping MICOS complex. How all these proteins work together to maintain the delicate equilibrium remains largely elusive. Recent advancements in super-resolution microscopy provide the necessary localization precision to investigate the distribution of single proteins and protein clusters within mammalian cells. To apply this to MERCS, new labeling strategies are necessary to harness the full potential of the techniques and to enable multiplexed imaging of these multi-protein micro-environments. Therefore, in the first part of this thesis I evaluated several multiplexing approaches for super-resolution microscopy. By implementing exchange DNA-PAINT for MINFLUX microscopy, I was able to record the distribution of three mitochondrial proteins, thereby increasing the multiplexing ability of MINFLUX. Next, I investigated an antibody-free labeling approach based on the heterodimeric interaction of small coiled-coil forming peptides. I found that these tags are generally suitable for labeling of abundant proteins in fixed cells. However, I discovered that the labeling efficiency is insufficient for super-resolution microscopy methods providing resolution at the scale of single proteins or protein clusters. In the second part of this thesis, I investigated the interconnection between the architecture of MERCS and mitochondrial function and ultrastructure. I identified MERCS-associated proteins that co-migrated with the mitochondrial bridging complex (MIB) in complexome profiling data and further investigated a subset of them, namely VAPB, BAP31, CISD2, CKAP4 and PDZD8. By performing loss-of-function experiments, I found that the mitochondrial calcium uptake was reduced upon depletion of VAPB, BAP31, CISD2 or CKAP4. The investigation of MERCS abundance and mitochondrial ultrastructure revealed a correlation between these two factors, both of which were altered in cells devoid of BAP31 or CISD2. Overall, my work demonstrates the link between MERCS and mitochondrial function and ultrastructure. I found that the roles of the many MERCS-associated proteins are interconnected. Correlating the effects of multiple proteins provided new insights into these important but still enigmatic contact sites.
Keywords: super-resolution microscopy; MERCS; Mitochondria-ER contact sites; MICOS complex; multiplexing