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

细胞的移动能力是发育、血管生成和损伤后受损组织修复的重要过程，需要制造新的蛋白质。为了使细胞能够做到这一点，细胞必须解码存储在遗传物质（DNA）基因序列中的关键信息，以便在正确的时间以正确的数量产生种类繁多的正确类型的基本蛋白质。从DNA到蛋白质的一般信息传递是由信使RNA （mRNA）完成的，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位被一个复杂的、高度调节的机器（称为核糖体）解码成蛋白质，这个过程被称为翻译。局部蛋白质合成允许细胞在细胞中需要蛋白质的地方和时间制造蛋白质，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，并使核糖体在细胞的最佳利益中发挥作用，这一机制需要相互作用的辅助蛋白（翻译起始因子；eIF），并确保mRNA和核糖体结合成一个高度调节的大型起始复合物，以制造所需的蛋白质。那么细胞是如何在正确的时间、正确的地点完成这个过程的呢？起始因子本身的相互作用是哺乳动物细胞调控的主要部位。4e结合蛋白1 （4E-BP1）阻止eIF4E与支架蛋白eIF4G的相互作用，并阻止mRNA向核糖体的募集并停止蛋白质合成。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。细胞通过磷酸基团标记4E-BP1， eIF4E， eIF4G和核糖体，这一过程被称为磷酸化。这种修饰促进4E-BP1从eIF4E/mRNA中释放，随后可以结合eIF4G并形成所需的多蛋白起始复合物，以制造所需的正确类型和数量的蛋白质。然而，我们仍然不知道细胞如何控制在迁移过程中的细胞中的局部蛋白质合成。通过使用专门的显微镜技术“观察”细胞内部，我们知道起始因子被分散地定位在细胞的特定区域；它们不只是漂浮着。在这里描述的工作中，我们想要研究成纤维细胞在哪里以及如何在它们被提示迁移时定位它们的翻译机制。然后，我们想要了解哪些信号是导致这种定位所必需的，并显示这些区域是否反映了细胞在培养中移动时产生蛋白质的活跃区域。这些研究将大大增加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了新的潜在途径。

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.