An optical method for detecting endoplasmic reticulum and mitochondrial associated membranes*

Mark Harmon, Mandy Jackson, Paul Skehel

Centre for Integrative Physiology and Euan MacDonald Centre for MND Research, University of Edinburgh

Interactions between the ER and mitochondrial membrane play a critical role in a variety of processes including calcium exchange, lipid metabolism and autophagy. Close apposition is achieved by the formation of protein complexes that tether the organelles at specialised junctions termed mitochondrial associated membranes (MAMs). The distance between the membranes at these junctions is heterogeneous and abnormal changes to ER-mitochondria contacts have been associated with neurodegeneration. Studies have shown that VAPB is enriched at these junctions and forms a complex with the outer mitochondrial membrane protein PTPIP51 to regulate ER-mitochondria interactions(1). A mis-sense point mutation VAPBP56S is associated with ALS8(2). It is suggested that the disease may result, in part, from mitochondrial dysfunction resulting from a failure to maintain functional MAMs.

Using split fluorescent venus proteins targeted to the ER and the outer mitochondrial membrane, we can visualize and detect changes to ER-mitochondria contact sites in living-cells and relate any changes to physiological responses such as calcium signalling and mitochondrial function. This also provides a platform for investigating the effect of VAPB mutations on the formation and regulation of MAMs.

The split venus expression constructs we have generated using the C-terminus of VAPB (Venus1-ER) and the outer mitochondrial membrane anchor of TOMM20 (Venus2-Mito) are correctly targeted to cellular membranes. Co-expression of these fusion proteins generates punctate fluorescent signals at regions of tight juxtaposition (<5nm) between ER and mitochondrial membranes, consistent with a MAM localisation. The induction of a serum deprivation stress causes a significant increase in the mean number of ER-mitochondria contacts per cell as previously reported in the literature(3).

These results suggest that our split venus reporter constructs will allow the detection and characterisation of MAMs in living cells. Further work will allow us to directly correlate the features of MAM with mitochondrial activity and the regulation of Ca2+ homeostasis. Furthermore, we may establish how mutations in proteins such as VAPB influence the activity of MAMs and how this might lead to neurodegeneration.

References

  1. Stoica R, De Vos KJ, Paillusson S et al. (2014) Nature Communications; 5:3996
  2. Nishimura AL, Mitne-Neto M, Silva HC et al.(2004) American Journal of Human Genetics; 75(5):822-31
  3. Csordás G, Renken C, Várnai P et al. (2006) The Journal of Cell Biology; 174(7):915-21

Funded by: This research is supported by a joint studentship between the University of Edinburgh and Zhejiang University.  The cost of printing the poster was kindly provided by the Euan MacDonald Centre.

* entered into the PhD student poster competition