Investigating reciprocal regulation of the cytoskeleton and protein translation machinery

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The cytoskeleton plays an essential role in the cell as a dynamic internal framework. It imparts cell shape, routeways for intra-cellular transport, physical support, and organisation of surface receptors and related signaling factors that are sensitive to external mechanical forces. Protein synthesis is a highly complex process involving the translation of mRNAs into polypeptides by massive molecular machines known as ribosomes. In addition, a suite of adaptor-like nucleic acids called tRNAs, and a host of different translation factors are required to coordinate the manufacture of new proteins. There is growing appreciation that these two cellular phenomena do not work in isolation but rather in concert. The cytoskeleton appears to provide a scaffold for the positioning of the protein translation machinery including eukaryotic initiation factors, ribosomes, mRNAs, and aminoacyl-tRNA synthetases. In return, some of these factors have been reported to play role in organising the actin protein filaments of the cytoskeleton. Further, biochemical disruption of this filamentous network has been shown to significantly interfere with protein synthesis. However, despite these connections the cross regulation of these two complex cellular machineries remains under explored. This project seeks to expand our knowledge of the inter-relationship between the cytoskeleton and protein translation. We will focus our attention on a key protein translation initiation factor that our preliminary data shows binds to the cytoskeleton. Importantly, when levels of this factor are significantly reduced in the cell using a technique called RNA interference (RNAi) we find there is a striking change in the behaviour of the cytoskeleton, but surprisingly without affecting the total levels of the proteins that make up the cytoskeleton such as actin. However, we find this translation factor is important for localised protein translation at key signaling hubs bound to the cytoskeleton when the cells are exposed to mechanical force. We will use a range of biological approaches including cell biology, mass spectrometry and the latest next-generation sequencing techniques to deconvolute how this protein translation factor regulates the cytoskeleton, and also modulates mechanically-induced protein synthesis. We believe this work will also shed light on general principles that explain how and why the cytoskeleton and protein translation machinery cross-regulate each other. In the long term, fundamental findings from this work may have implications for the treatment of diseases including nervous system disorders and cancer.