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

细胞的活动能力是损伤后发育、血管生成和受损组织修复的重要过程，需要制造新的蛋白质。为了细胞能够做到这一点，储存在遗传物质(DNA)的基因序列中的关键信息必须由细胞解码，以在正确的时间、在正确的数量中产生各种正确类型的必要蛋白质。信息从DNA到蛋白质的一般传递是由信使RNA(信使RNA)执行的，信使RNA是DNA序列的副本。当需要时，这种mRNA必须在细胞的不同部分被称为核糖体的复杂、高度调控的机器解码为蛋白质，这一过程被称为翻译。局部蛋白质合成允许细胞在细胞需要的地方和时间准确地制造蛋白质，而不必浪费时间和精力将蛋白质移动到正确的位置。为了高效、准确地工作，并使核糖体以细胞的最佳利益发挥作用，这一机制需要相互作用的辅助蛋白(翻译启动因子；EIF)，并确保mRNA和核糖体聚集在一起形成高度调控的大型启动复合体，以产生所需的蛋白质。那么，细胞如何在正确的时间、正确的地点实现这一点呢？启动因子本身的相互作用是哺乳动物细胞中调节的主要部位。一种名为4E-结合蛋白1(4E-BP1)的蛋白质阻止eIF4E与支架蛋白eIF4G的相互作用，阻止mRNA募集到核糖体并停止蛋白质合成。当需要蛋白质合成时，细胞向4E-BP1发出信号，从4E-BP1/eIF4E/mRNA复合体中释放eIF4E/mRNA，使其发挥作用。细胞通过在被称为磷酸化的过程中用磷酸基团标记4E-BP1、eIF4E、eIF4G和核糖体来做到这一点。这种修饰促进了4E-BP1从eIF4E/mRNA中释放出来，随后可以与eIF4G结合，形成产生正确类型和数量所需蛋白质所需的多蛋白质起始复合体。然而，我们仍然不知道细胞如何控制处于迁移过程中的细胞的局部蛋白质合成。通过使用专门的显微镜技术观察细胞内部，我们知道启动因子分散地定位于细胞中的特定区域；它们不仅仅是漂浮在细胞内。在这里描述的工作中，我们想要调查成纤维细胞在被提示迁移时，它们在哪里以及如何定位它们的翻译机制。然后，我们想了解哪些信号是实现这种定位所必需的，并表明这些区域是否反映了细胞在培养过程中移动时产生蛋白质的活跃区域。这些研究将大大提高我们对控制蛋白质合成在调节细胞生长和迁移中的重要性的总体理解，为控制已经获得在体内移动能力的癌细胞开辟新的潜在途径。

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.