Endoplasmic reticulum-mitochondria contacts: molecular hubs for neuronal regulation
Final Report Abstract
Understanding how ER contributes to mitochondrial movement would be a great contribution into understanding the behavior of that organelle. Given mitochondria involvement in many of the neurodegenerative disorders this study had the potential to contribute to the development of neurodegenerative phenotypes. So far majority of the studies of contacts was done using biochemical and histological approaches and fixed tissues without the possibility to monitor contacts in living cells. Given the lack of previous studies, I set up to test which of the available techniques would be the most suitable for answering question at hand. I tested two microscopy techniques FRET and FLIM and assessed their appropriateness for this study. While FRET proved to be not sensitive enough to detect contacts and results were inconclusive, FLIM showed a potential for working effectively when used in combination with COS7 cells. Combined with analysis algorithms and developed post-processing protocols, it had a potential for serving the project well. Unfortunately, the same was not the case when the experiments were done in primary neuronal cultures. Taken together, I can conclude that currently available methodology does not allow for reliable and convincing visualization of the ER-mitochondria contacts in living cells. It is likely due to the low resolution of the used techniques, high demand of the signal needed for proper calculation of FLIM signals. In support of the low resolution is a study by Cui-Wang et al. 2012 showing how dense is neuronal ER network. I strongly believe that in the future new advances in super resolution microscopy will allow for monitoring dynamics of those contacts in living cells, and therefore answer questions related to ER and mitochondrial biology.
