Harnessing protein unfolding and aggregation in mechanotransduction

机械力塑造了我们身体的发育和功能。例如，当我们的肌肉以更大的力量扩张和收缩时，一种机制会感知到这些力量，并加强肌肉末端与肌腱的连接，使它们足够强壮，能够承受增加的力量。这个过程被称为机械转导，它是我们身体许多功能的核心。提出的研究重点是涉及细胞如何感知这些机械力的分子机制。人体内的所有细胞都通过与邻近细胞的黏附，以及与细胞周围的密集蛋白质网（细胞外基质）的黏附，保持在正确的位置。细胞通过一种叫做整合素的细胞表面蛋白质附着在这种基质上。Talin是将整合素连接到细胞力产生机制的主要连接蛋白，在一端接合整合素并将其连接到细胞骨架上。当细胞骨架拉紧整合素锚点时，talin像弹簧一样伸展，talin的展开会招募到蛋白质vinculin，这以一种力依赖的方式加强了粘附。虽然这种模式提供了一种可行的力诱导化学变化的机制，即细胞内血管蛋白的重新分配，但它也提出了许多问题，这些问题是本研究的重点。在这个建议中，我们建立在我们最近发现的机械转导如何工作的两个新的和意想不到的谜团的基础上。我们已经发现，talin展开可以导致talin分子通过一个称为聚集的过程进行自组装。这是一个意想不到的发现，因为蛋白质聚集体以其在疾病中的作用而闻名，例如痴呆症和阿尔茨海默病都是由蛋白质聚集体的积累引起的。我们的细胞利用“伴侣”蛋白来保护自己免受这种聚集体的侵害，这种蛋白可以溶解并重新折叠错误折叠的蛋白质。我们的中心假设是，这两个有害的过程，蛋白质展开和蛋白质聚集，已经被细胞利用，作为优雅的力感知机制，使细胞能够感知力并将其转化为生物信号。我们想要验证的假设是，锚位点的一个正常特征是由拉伸的talin分子形成的网状结构，这为整合素粘附所需的许多附加组分的组装提供了坚实的平台。我们的试验数据表明，这种网络的形成和重排涉及到特定的伴侣来控制这一过程，并确保它不会出错。我们将结合两个实验室的专业知识来验证这一假设。Goult实验室将使用生化、生物物理和结构方法来描述这些成分是如何协同工作的，并确定可以对分子进行特定的改变以改变它们的活性。布朗实验室将利用强大的遗传学和成像方法，这些方法可用于果蝇，以测试在生物体内需要整合素机制的不同过程中talin网的形成和重塑的重要性，例如肌肉的附着和干细胞的锚定。这项研究在几个层面上都很重要。我们的发现将提高我们对力如何加强细胞粘附，以及如何避免病理性蛋白质聚集的理解，对理解人类疾病有潜在的好处。由细胞粘附减弱引起的疾病可以通过模拟力信号和增强粘附的干预措施得到改善。同样，癌细胞的移动或转移使癌症更难治疗，而加强黏附将固定癌细胞并抑制细胞移动。果蝇的实验优势将使我们能够研究生物体在其整个生命周期中特定蛋白质-蛋白质相互作用的作用，然后将这些知识应用于人类。

Mechanical forces shape how our bodies develop and function. For instance as our muscles enlarge and contract with greater force, a mechanism senses these forces and strengthens the attachment of muscle ends to tendons so they are strong enough to withstand the increased force. This process is called mechanotransduction and it is central to many of our body's functions. The proposed research focuses on the molecular machinery involved in how cells sense these mechanical forces.All cells in the human body are held in the correct place via adhesion to neighbouring cells, and to a dense meshwork of proteins that surround cells, the extracellular matrix. Cells adhere to this matrix via cell surface proteins called integrins. Talin is the main linker protein coupling integrins to the cell's force generating machinery, engaging integrin at one end and coupling it to the cytoskeleton. As the cytoskeleton pulls on the integrin anchors, talin stretches like a spring and unfolding of talin recruits the protein vinculin, which reinforces the adhesion in a force-dependent manner. While this paradigm provides a feasible mechanism for force to induce a chemical change, namely the redistribution of vinculin within the cell, it also raises many questions, which are the focus of this research. In this proposal we build on our recent discovery of two new and unexpected pieces of the puzzle of how mechanotransduction works. We have discovered that talin unfolding can lead to self-assembly of talin molecules by a process called aggregation. This is an unexpected discovery, as protein aggregates are best known for their role in disease, for instance dementia and Alzheimer's disease are both caused by accumulation of protein aggregates. Our cells protect themselves from such aggregates using "chaperone" proteins that dissolve and refold misfolded proteins. Our central hypothesis is that these two harmful processes, protein unfolding and protein aggregation, have been harnessed by the cell to serve as elegant force sensing mechanisms that enable the cell to sense forces and convert them into biological signals. The hypothesis that we would like to test is that a normal feature of anchor sites is the formation of a meshwork of stretched talin molecules, which provide a solid platform for the assembly of many additional components required for integrin adhesion. Our pilot data suggest that the formation and rearrangement of this meshwork involves specific chaperones to control this process and to ensure it does not go wrong. We will test this hypothesis by combining the expertise of our two labs. The Goult lab will use biochemical, biophysical and structural methods to characterize how the components work together, and to identify specific changes that can be made to the molecules to alter their activity. The Brown lab will exploit the powerful genetics and imaging approaches that can be used in the fruit fly Drosophila to test the importance of the formation and remodelling of the talin meshwork in different processes that require the integrin machinery within the organism, such as attachment of muscles and anchoring of stem cells.This research is important at several levels. Our discoveries will improve our understanding of how forces strengthen cell adhesion, and how pathological protein aggregation is avoided, with potential benefits to the understanding of human disease. Diseases caused by weakening of cell adhesion may be improved by interventions that mimic the force signal and strengthen adhesion. Similarly, movement of cancer cells, or metastasis, renders cancers much more difficult to treat, and strengthening adhesion will anchor cancer cells and restrain cell movement. The experimental advantages of Drosophila will allow us to investigate the role of specific protein-protein interactions within an organism throughout its life cycle, and this knowledge will then be applied to humans.