Localised protein synthesis in fibroblasts during cell spreading and migration in 3D culture

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The ability of cells to move about is an important process in development, generation of blood vessels and repair of damaged tissues after injury, requiring new proteins to be made. For cells to be able to do this, critical information stored in the gene sequences of the genetic material (DNA) has to be decoded by the cell to produce a wide variety of essential proteins of the right type, in the right amount and at the right time. The general transfer of information from DNA to protein is carried out by the messenger RNA (mRNA), which is a copy of the DNA sequence. When required, this mRNA has to be decoded into protein in different parts of the cell by a complex, highly regulated machine termed a ribosome, in a process known as translation. Localised protein synthesis allows the cell to make the protein exactly where and when it needs it in the cell without having to waste time and energy moving the protein around to the correct location. To work efficiently, accurately, and to allow the ribosome to function in the best interests of the cell, this machinery requires helper proteins (translation initiation factors; eIF) that interact with each other, and also make sure that the mRNA and the ribosome come together into a highly regulated, large initiation complex to make the proteins required. So how does the cell make this happen in the right place at the right time? The interaction of the initiation factors themselves is a major site for regulation in mammalian cells. One protein, 4E-binding protein 1 (4E-BP1) prevents the interaction of eIF4E with the scaffold protein, eIF4G, and stops the recruitment of mRNA to the ribosome and halts protein synthesis. When protein synthesis is needed, the cell signals to 4E-BP1 to release the eIF4E/mRNA from the 4E-BP1/eIF4E/mRNA complex to let it work. The cell does this by marking the 4E-BP1, eIF4E, eIF4G and ribosomes with phosphate groups in a process known as phosphorylation. This modification promotes 4E-BP1 release from eIF4E/mRNA which can subsequently bind to eIF4G and form the multi-protein initiation complex required to make the correct types and amounts of protein needed. However, we still do not know how the cell controls localised protein synthesis in cells which are in the process of migrating. From 'looking' inside the cell with specialised microscopy techniques, we know that the initiation factors are discretely localised to specific regions in the cell; they are not just floating about. In the work described here we want to investigate where and how fibroblasts cells localise their translational machinery when they are prompted to migrate. We then want to understand which signals are required to bring about this localisation and show whether these regions reflect active areas where proteins are being made as the cell moves about in culture. These studies will substantially increase our general understanding of the significance of the control of protein synthesis in the regulation of cell growth and migration, opening up new potential avenues for controlling cancer cells which have acquired the ability to move about the body.