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

3D 培养中细胞扩散和迁移过程中成纤维细胞的局部蛋白质合成

• 批准号: BB/H018956/1
• 负责人: Simon Morley
• 金额: $ 41.91万
• 依托单位: University of Sussex
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH018956%2F1
• 关键词: Localised; protein; synthesis; fibroblasts; during

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.

细胞移动的能力是发育、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，储存在遗传物质（DNA）基因序列中的关键信息必须由细胞解码，以在正确的时间以正确的数量产生各种各样的正确类型的必需蛋白质。信息从DNA到蛋白质的一般传递是由信使RNA（mRNA）完成的，它是DNA序列的一个拷贝。当需要时，这种mRNA必须在细胞的不同部位被称为核糖体的复杂、高度调节的机器解码成蛋白质，这一过程称为翻译。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精确地制造蛋白质，而不必浪费时间和能量将蛋白质移动到正确的位置。为了有效、准确地工作，并使核糖体以细胞的最佳利益发挥作用，这种机制需要相互作用的辅助蛋白（翻译起始因子; eIF），并确保mRNA和核糖体一起形成高度调节的大起始复合物，以产生所需的蛋白质。那么，细胞是如何在正确的时间在正确的地点发生这种情况的呢？起始因子本身的相互作用是哺乳动物细胞中调节的主要位点。一种蛋白质，4 E-结合蛋白1（4 E-BP 1），阻止eIF 4 E与支架蛋白eIF 4G的相互作用，并停止mRNA向核糖体的募集和停止蛋白质合成。当需要蛋白质合成时，细胞向4 E-BP 1发出信号，从4 E-BP 1/eIF 4 E/mRNA复合物中释放eIF 4 E/mRNA，让其发挥作用。细胞通过在称为磷酸化的过程中用磷酸基团标记4 E-BP 1，eIF 4 E，eIF 4G和核糖体来做到这一点。这种修饰促进4 E-BP 1从eIF 4 E/mRNA释放，其随后可以结合eIF 4G并形成制备所需的正确类型和量的蛋白质所需的多蛋白质起始复合物。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，我们知道起始因子分散地定位于细胞中的特定区域;它们不仅仅是漂浮在周围。在这里描述的工作中，我们想研究成纤维细胞在被促使迁移时，在哪里以及如何定位它们的翻译机器。然后，我们想了解需要哪些信号来实现这种定位，并显示这些区域是否反映了细胞在培养中移动时蛋白质生成的活跃区域。这些研究将大大增加我们对蛋白质合成控制在细胞生长和迁移调节中的重要性的一般理解，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。

项目成果

Translation initiation factors and active sites of protein synthesis co-localize at the leading edge of migrating fibroblasts

翻译起始因子和蛋白质合成活性位点共定位于迁移成纤维细胞的前缘

• DOI: 10.1042/bj20110435
• 发表时间: 2011
• 期刊: Biochemical Journal
• 影响因子: 4.1
• 作者: Willett M

Murine norovirus 1 (MNV1) replication induces translational control of the host by regulating eIF4E activity during infection.

• DOI: 10.1074/jbc.m114.602649
• 发表时间: 2015-02-20
• 期刊: The Journal of biological chemistry
• 作者: Royall E;Doyle N;Abdul-Wahab A;Emmott E;Morley SJ;Goodfellow I;Roberts LO;Locker N
• 通讯作者: Locker N

Sumoylation of eIF4A2 affects stress granule formation.

• DOI: 10.1242/jcs.184614
• 发表时间: 2016-06-15
• 期刊: Journal of cell science
• 影响因子: 4
• 作者: Jongjitwimol J;Baldock RA;Morley SJ;Watts FZ
• 通讯作者: Watts FZ

The helicase, DDX3X, interacts with poly(A)-binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading.

解旋酶DDX3X在迁移成纤维细胞的前沿与poly（a）结合蛋白1（PABP1）和Caprin-1相互作用，对于有效的细胞扩散是必需的。

• DOI: 10.1042/bcj20170354
• 发表时间: 2017-08-30
• 期刊: The Biochemical journal
• 作者: Copsey AC;Cooper S;Parker R;Lineham E;Lapworth C;Jallad D;Sweet S;Morley SJ
• 通讯作者: Morley SJ