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

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.

Localised protein synthesis coupled to mRNA targeting spatially restricts the synthesis of specific proteins required for cell spreading and migration but little is known about how the cell spatially regulates the translational machinery. With MRC5 fibroblasts in 2D culture, we have used scanning confocal microscopy and immunofluorescence to show an ECM-dependent enrichment of the translational machinery in lamellipodia and association with integrins at focal adhesions at the leading edges of spreading and migrating cells. eIF4E co-fractionates with filamentous tubulin and vesicles which decorate microtubules in the perinuclear region and lamellipodia. These vesicles exhibit a staining pattern similar to the Golgi-enriched C5 ceramide suggesting a role for the Golgi in localising eIF4E at the leading edge of spreading and migrating cells. However, cells in 2D culture show morphology with little in common with cells in vivo. When cultured in ECM-coated 3D matrices, such fibroblasts are bipolar or spindle-shaped, often lack discrete focal contacts and stress fibres and show typical mesenchymal migration along the fibres, making them ideal in vivo model system to investigate localised translation during spreading and migration. To understand the compartmentalisation of the translation machinery during cell migration, we will use immunofluorescence to monitor initiation factor and ribosome localisation in MRC5 fibroblasts showing distinct cellular morphologies. This will be complemented by replating cells from 2D to 3D culture or following stimulation with PDGF or LPA to allow us to correlate localisation with cell morphology and general translation rates. We will use cell-permeable inhibitors and RNAi technology to investigate the mechanisms and signalling pathways responsible for ECM- and growth factor-mediated localisation of the translational machinery and employ novel technology to determine how such treatments promote localised, active translation.

The main beneficiaries here will be research colleagues and those in the Pharma industry with an interest in cell motility and migration during growth, wound healing and metastasis. In addition to presentation of this 'basic, blue-sky research' at international meetings, the main impact will be on: New Collaborations; As part of this proposal, we will be starting a new collaboration with Stefan Przyborski at Durham University (and Reinnervate Ltd) in the use of 3D polystyrene matrices in the study of signaling and translational control in non-transformed and transformed fibroblasts. Also, we will initiate a collaboration with Jonathan Yewdell (NIH) to pioneer the visualization of localized protein synthesis in spreading and migrating cells in 2D and 3D culture. Regarding the inhibitors of mTORC1 signalling (RAD001, Torin 1), we will continue to interact with Novartis (Basel) and the Whithead Institute, USA. Our preliminary data associated with this application for funding used both of these drugs. In future, we will build upon these collaborations to secure access to the next generation of signalling inhibitors which are currently entering clinical trials. This work will also help foster new investigations into how these signalling pathways impinge on wound healing. This has promoted a new BBSRC-funded collaboration with Dr. Sarah Newbury, research faculty at the Medical School at Sussex (BSMS), to look at the role of phosphorylation events in wound healing using Drosophila as a model system. We have already secured pilot funding with Dr.Tim Chevassut, a BSMS clinician, to investigate the differential sensitivity of leukaemic stem cells to our signalling inhibitors. With an MRC Senior Clinical Research Fellow at BSMS, Dr. Anthony Chalmers, we have started collaborating to investigate whether RAD001 and Torin 1 can sensitise human glioma tumour cells to radiotherapy. Skills, training and knowledge economy; The PDRA will have to master a number of timely and intensive techniques and approaches, optimising them accordingly. This will provide a fertile environment for training and development of new skills. The characterisation of growth conditions how this relates to our model systems for studying protein synthesis will expand our way of thinking about translational regulation in general and how mRNAs are triaged by the cells. New networking groups; South Coast RNA Group. As part of an efficient vehicle for efficient dissemination of our work, with Dr. Sarah Newbury and funded by Sussex IP, we have started a group of like-minded workers who will meet every quarter at various locations on the south coast. The format will be seminars and discussions to promote interactions and collaborations; the first meeting will be in November 2009 at Sussex. Sussex Cancer Group, is a group of researchers and clinicians at Sussex working in themes of cancer, growth control, patient care and clinical trials, brought together by Dr. Chalmers. This group meets 4 times per year and is funded by Sussex IP and Industrial sponsors. This networking opportunity has led to a number of fruitful discussions and the collaborations outlined in above and our proposed work in cell already has a lot of interest as a model for metastasis using patient material . Schools and public engagement; As part of our current lab remit to publicise our work, I have talked at a local girl's 6th form college and we will have 6th form students carrying out 'hands-on' projects in the lab again over the summer. Both my PhD student and myself have interacted with 8-14yr olds at the Brighton Science Fair in the form of 'hands-on' playdough model sculpturing of cells, their structures and morphology. I have also participated in discussions about the use of animal research in science at a public forum in Brighton and regularly attend local Café Scientifique presentations to talk about what we do in the lab.