A new role for intermediate filaments in the secretory pathway

中间丝在分泌途径中的新作用

• 批准号: BB/T000945/1
• 负责人: Martin Lowe
• 金额: $ 61.31万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT000945%2F1
• 关键词: new; role; intermediate; filaments; secretory

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.

蛋白质是我们细胞内的主要组成部分，也从我们的细胞中分泌出来，以介导细胞间通讯，并产生细胞在我们体内附着的细胞外基质。这种基质对于组织的形成和维持至关重要，并提供机械强度，这在骨骼和肌腱中尤其明显。因此，细胞通过一个可以根据各种外部影响（包括细胞的机械环境）进行调节的过程不断地制造和分泌蛋白质。大约三分之一的细胞蛋白质，包括那些分泌到细胞外部的蛋白质，是在分泌途径内产生的。该途径由不同的区室组成，货物蛋白通过这些区室顺序运输，并在此过程中接受功能上重要的修饰。分泌通路中心的隔室是高尔基体，它是运输的枢纽，也​​是货物修饰的主要场所。分泌途径的组织和蛋白质在分泌室（包括高尔基体）之间的运动取决于纤维网络，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，凸显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 的高尔基体驻留蛋白，其突变会导致人类皮肤和骨骼疾病。我们已经证明 GORAB 在高尔基体膜上形成称为结构域的离散结构，该过程对于高尔基体的货物修饰很重要，并且它的损失会影响细胞外基质的组装。最近，我们发现 GORAB 和一种称为细胞质中间丝的细胞骨架结构之间存在新的相互作用。尽管中间丝经过充分研究并且已知对细胞完整性很重要，但我们对它们如何影响分泌途径的组织或功能知之甚少。我们对高尔基体与中间丝的新关联的鉴定表明这种相互作用在分泌途径组织和功能中发挥着重要作用。在本提案中，我们的目标是确定 GORAB 如何在高尔基膜上形成结构域、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。然后一个主要目标是确定 GORAB-中间丝相互作用的功能意义，测试我们的假设，即它对高尔基体组织和功能很重要，并通过机械实验来确定这是如何发生的。另一个关键方面是确定分泌途径，特别是高尔基体如何响应细胞机械环境的变化，这对于分泌途径内蛋白质的正确生产至关重要。由于细胞质中间丝在细胞内发挥着重要的机械作用，我们将测试它们将细胞机械环境的变化与高尔基体的功能组织直接耦合的程度，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如何组织、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耦合非常重要，所有这些对于人类健康都至关重要。

项目成果

Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex.

• DOI: 10.1242/jcs.260577
• 发表时间: 2023-10-15
• 期刊: JOURNAL OF CELL SCIENCE
• 影响因子: 4
• 作者: Vitali, Teresa;Sanchez-Alvarez, Rosa;Witkos, Tomasz M.;Bantounas, Ioannis;Cutiongco, Marie F. A.;Dudek, Michal;Yan, Guanhua;Mironov, Alexander A.;Swift, Joe;Lowe, Martin
• 通讯作者: Lowe, Martin

Immunofluorescence Microscopy of the Mammalian Golgi Apparatus.

哺乳动物高尔基体的免疫荧光显微镜。

• DOI: 10.1007/978-1-0716-2639-9_8
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Arab M

GOLPH3 keeps the Golgi residents at home.

• DOI: 10.1083/jcb.202108147
• 发表时间: 2021-10-04
• 期刊: The Journal of cell biology
• 作者: Lowe M