A new role for intermediate filaments in the secretory pathway

蛋白质是我们细胞内的主要组成部分，也从我们的细胞分泌来介导细胞间的通讯，以及产生细胞附着在我们体内的细胞外基质。这种基质对于组织形成和维持至关重要，并提供机械强度，这在骨和肌腱中尤其明显。因此，细胞通过一个可以响应各种外部影响（包括细胞的机械环境）进行调节的过程不断制造和分泌蛋白质。大约三分之一的细胞蛋白质，包括分泌到细胞外部的蛋白质，是在分泌途径中产生的。该途径由货物蛋白依次转运的各个隔室组成，在转运过程中接受功能上重要的修饰。位于分泌途径中心的隔室是高尔基体，其充当运输枢纽，以及作为货物修饰的主要位点。分泌途径的组织和蛋白质在分泌区室（包括高尔基体）之间的运动依赖于纤维网络，通常称为细胞骨架。分泌途径功能和/或细胞骨架组织的缺陷可能导致疾病，突出了这些过程对人类健康的重要性。这项研究的重点是一种名为GORAB的高尔基体常驻蛋白，它的突变会导致人类皮肤和骨骼疾病。我们已经表明，GORAB形成离散的结构称为域在高尔基体膜，这一过程是重要的货物修改在高尔基体，它的损失影响组装的细胞外基质。最近，我们发现了GORAB和一种称为细胞质中间丝的细胞骨架结构之间的新型相互作用。虽然中间丝是非常好的研究和已知的是重要的细胞的完整性，我们知道很少关于他们如何可能影响组织或功能的分泌途径。我们确定了一个新的协会的高尔基体与中间丝这一相互作用在分泌途径的组织和功能的重要作用。在这个建议中，我们的目标是确定GORAB如何在高尔基体膜上形成结构域，如何控制结构域的组装，以及结构域如何与细胞质中间丝结合。一个主要的目标是，然后确定GORAB中间丝相互作用的功能意义，测试我们的假设，这是重要的高尔基体的组织和功能，与机械实验，以确定这是如何发生的。另一个关键方面将是确定分泌途径，特别是高尔基体，如何响应细胞机械环境的变化，这对分泌途径内蛋白质的正确产生至关重要。由于细胞质中间丝在细胞内发挥着重要的机械作用，我们将测试它们在多大程度上直接将细胞机械环境的变化与高尔基体的功能组织相结合，重点关注与GORAB的相互作用，但也探索其他可能的机制。这项工作对于确定分泌途径是如何组织的，中间丝如何与该途径相互作用，以及分泌途径如何与其机械环境相结合非常重要，所有这些都对人类健康至关重要。

Proteins are major building blocks within our cells, and are also secreted from our cells to mediate intercellular communication, as well as generating the extracellular matrix to which cells attach within our bodies. This matrix is vital for tissue formation and maintenance, and provides mechanical strength, which is especially apparent in bone and tendon. Cells therefore constantly make and secrete proteins via a process that can be regulated in response to various external influences, including the mechanical environment of the cell. Approximately one third of all cellular proteins, including those proteins secreted to the cell exterior, are made within the secretory pathway. This pathway is comprised of various compartments through which cargo proteins are sequentially transported, receiving functionally important modifications as they do so. The compartment at the centre of the secretory pathway is the Golgi apparatus, which serves as a hub for transport, as well as being a major site of cargo modification. The organization of the secretory pathway and movement of proteins between secretory compartments, including the Golgi apparatus, is dependent upon a fibrous network, generally referred to as the cytoskeleton. Defects in secretory pathway function and/or cytoskeleton organization can cause disease, highlighting the importance of these processes for human health. The focus of this study is a Golgi resident protein called GORAB, whose mutation causes a skin and bone disorder in humans. We have shown that GORAB forms discrete structures called domains at the Golgi membrane, that this process is important for cargo modification at the Golgi, and that its loss affects assembly of the extracellular matrix. More recently, we have found a novel interaction between GORAB and a type of cytoskeletal structure called cytoplasmic intermediate filaments. Although intermediate filaments are very well studied and known to be important for cell integrity, we know very little about how they might affect the organization or function of the secretory pathway. Our identification of a novel association of the Golgi apparatus with intermediate filaments suggests an important role for this interaction in secretory pathway organization and function. In this proposal we aim to identify how GORAB forms domains at the Golgi membrane, how the assembly of domains is controlled, and how the domains bind to cytoplasmic intermediate filaments. A major goal is then to determine the functional significance of the GORAB-intermediate filament interaction, testing our hypothesis that it is important for Golgi organization and function, with mechanistic experiments to determine how this occurs. Another key aspect will be to determine how the secretory pathway, and the Golgi apparatus in particular, responds to changes in the mechanical environment of the cell, which is vital for the correct production of proteins within the secretory pathway. Because cytoplasmic intermediate filaments play an important mechanical role within cells, we will test the extent to which they directly couple changes in the mechanical environment of cells to the functional organization of the Golgi apparatus, focusing on the interaction with GORAB, but also exploring other possible mechanisms at play. The work will be important for determining how the secretory pathway is organized, how intermediate filaments interact with this pathway, and how the secretory pathway is coupled to its mechanical environment, all of which are fundamentally important for human health.