A new role for intermediate filaments in the secretory pathway

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.

The secretory pathway is fundamentally important, producing ~30% of all cellular proteins. The Golgi apparatus lies at the heart of this pathway, acting as a trafficking hub and major site of cargo modification. The organization of the secretory pathway is dictated by the cytoskeleton, with well-established roles for microtubules and actin. However, the extent to which intermediate filaments contribute to secretory pathway organization or function remains to be determined. We have recently discovered a novel association of cytoplasmic intermediate filaments with the Golgi apparatus, which is mediated by an interaction between the Golgi resident protein GORAB, and vimentin. GORAB forms discrete domains at the Golgi membrane, which physically associate with vimentin intermediate filaments. We hypothesize that this association is required for the optimum organization and functionality of the Golgi apparatus., and We also propose that this interaction is important for mechanotransduction by directly linking cytoplasmic intermediate filaments to the Golgi apparatus. This would allow for changes in secretory pathway function in response to changes in cell mechanics, which in turn is dictated by the cellular environment. This project will test these hypotheses by using a combination of biochemistry, biophysics and cell biology. We will investigate how the GORAB domains are assembled, how their assembly is controlled, and how the interaction with cytoplasmic intermediate filaments is mediated. We will then determine the functional importance of this interaction, examining its role in Golgi organization and trafficking. We will also investigate how the Golgi apparatus responds to changes in cell mechanics, examining the role of GORAB and intermediate filaments in this process. Together, the findings will provide new insight into how the secretory pathway is organized, and how it is coupled to cell mechanics, as well as uncovering a new role for intermediate filaments.

Wherever possible we will try to maximize the impact of our research. We will adopt several strategies to achieve this, as indicated below.

Public engagement
We envisage that findings of our current study will be of interest to the public at large given the importance of the processes we will investigate to human health. We will communicate our findings to the public via the open access University website and through the Faculty of Biology, Medicine and Health (FBMH) research brochure. We will also notify the dedicated Faculty press officers of our findings at around the point of publication in academic journals. The press officer will then contact local and national news agencies, and prepare press releases that these agencies can use. The ML, CB, JS and TW labs have participated in several public engagement initiatives and we plan for this to continue. This will include hosting 2 A level students as part of the Nuffield bursary scheme. Members of the aforementioned labs have also participated in activities at local Science Fairs, and again, we plan for this to continue should the application be successful.

Exploitation and application
The work outlined in the proposal is basic research. Thus it is not trivial to realize the short and long term benefit of the work in terms of direct commercial or clinical exploitation. However, the scientific data obtained will increase the knowledge of UK and international scientists in the academic, clinical and commercial sectors. This contribution to knowledge will lead to improved understanding at the level of basic understanding of cellular function, and will also contribute towards an improved understanding of mechanisms of disease and ageing. This increase in knowledge will aid the design of better diagnosis and treatments for patients. This is particularly relevant to gerodermia osteodysplastica, but should also be applicable to other diseases associated with extracellular matrix defects, and more generally to ageing. Finally, because most biologics are synthesized in the secretory pathway, our findings may be exploited by the bio-pharmaceutical industry.

Training
The technical and intellectual knowledge acquired by the post-doctoral researcher during the course of the proposed work will equip this person to pursue a future career as an independent scientist. The interdisciplinary nature of this project will allow the PDRA to expand their expertise and equip them for a future research career, possibly at the interface of biology and physics. The researcher will also learn transferable skills that will increase their employability. Such highly trained individuals will contribute to the UK knowledge and skill base and ultimately the UK economy.

Impact deliverables and milestones
Key milestones will be the publication of research articles in leading open-access scientific journals, allowing us to maximize the exposure and thus impact of the work. We hope to publish papers following years 2 and 3 of the project. All raw data will be made available to other researchers at the point of publication. The findings will also be exposed to the research community through presentation at scientific conferences within the UK and abroad. The public view of the project, and any findings arising during it, will be maintained though our University website. This will allow us to publicize new information to scientific researchers, students, commercial companies, and the public at large.