The effect of elongation factor 1 alpha eEF1A on the organisation of polymerised actin filaments


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Annette Doyle1, Mark Murphy1, Mark Murphy1, R Crosby1
1School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, United Kingdom

Background

Many changes to the integral structure of the cell, the cytoskeleton occur in cancer cells. We are interested in the actin cytoskeleton and how its structure and organisation is affected by over-expression of the protein Elongation factor 1 alpha (eEF1A). eEF1A is over-expressed in 60% of breast cancers and 30% of ovarian tumours. It is suggested that eEF1A causes actin filaments to bundle causing profound effects on cellular function and cellular pathology. We therefore investigated the role of eEF1A on purified actin.

Method

As eEF1A sequence homology is highly conserved between species we are investigating whether yeast (yeEF1A) and human isoforms, heEF1A1 and heEF1A2 also reorganise actin filaments in vitro. For this study we are using purified actin from rabbit muscle and purified eEF1A protein from yeast and also both heEF1A1 and heEF1A2. In order to assess how yeEF1A interacts with actin, and the extent of this association/bundling, we are using atomic force, confocal and transmission electron microscopy to image polymerised actin with and without the yeast and human eEF1As. We are also using absorption spectroscopy to measure the rate of bundling and polymerisation of actin in real-time.

Results

Our initial data shows that the formation of F-actin bundles is facilitated in the presence of yeEF1A. Using absorption spectroscopy we found that the rate of actin polymerisation is increased in the presence of yeEF1A. Our data also suggests that yeEF1A effects the dynamics of F-actin polymerisation/depolymerisation with yeEF1A seemingly having a stabilising effect on F-actin.

Conclusion

With eEF1A found to be over-expressed in many cancers, we have recently shown that transformed human fibroblast cells (MRC-5-SV2), with characteristics of cancer cells and are known to over express eEF1A, are mechanically ‘weaker’ than their normal counter parts (MRC-5 cells). A possible explanation could be that the over-expression of eEF1A contributes to a reorganisation of actin.