Organization of the early secretory pathway in vertebrates: the role of the Mia gene family.

The internal organization of every cell in our bodies is inherently linked to their function. We study the way in which the individual compartments (organelles) within cells relate to one another in the context of protein secretion. This is fundamental for many aspects of life in particular the formation and maintenance of the extracellular matrix that surrounds cells in every tissue. This matrix is largely made up of collagen, the most abundant protein in our bodies. We have discovered that the way in which these organelles relate to one another requires specific proteins that were previously considered to have quite specific roles in collagen transport. We are now in a position to exploit the latest advances in genome engineering to define the role of a family of proteins (called the Mia family) in cell and tissue function. We can only develop this work so far in cells in culture and so also plan to develop our experiments using zebrafish models. This gives us huge advantages of being able to look at cells in context, as they interact and move within a living organism. Zebrafish are well established as a developmental model and many tools that are well suited to this project are already available. Fortunately for us, we have established a collaboration with Brian Link (Milwaukee, USA) who has recently developed some Mia gene family knockout fish.

While our work is very much aimed at defining the fundamental cell biology of this system it is also of significant relevance to society. Collagen is the most abundant protein in the human body, forming a vital protein scaffold to support cells and maintain tissue integrity. It is a critical component of cartilage and bone. Understanding the biology of the collagen matrix is fundamental to human health. As we age, loss of skin elasticity, poor wound healing, and an increased susceptibility to osteoarthritis and bone fractures become prevalent and the underlying cause is usually a reduction in the quality of collagen in the affected tissues. There are no effective treatments for many of these diseases. Conversely, abnormal accumulation of collagen causes fibrosis, a type of scarring, which is associated with 45% of all deaths (including those from cancer and cardiovascular disease). Here, we propose a project to define the fundamental mechanisms of protein secretion by looking at a holistic level at the early secretory pathway. We will however maintain an overall vision of considering the assembly and secretion of the collagen-rich extracellular matrix from cells. Any future opportunities to modulate tissue maintenance, and turnover will require a detailed understanding of the mechanisms by which collagen is made. The process is unfortunately tremendously complex, and we have only just begun to understand which components are involved in the system and what they do. Much work to date has been necessarily limited to simple systems. Our work will provide new insight into how cells are organized, how the system functions in the normal state, what goes wrong in particular human genetic diseases that affects these specific components, and into how collagen is synthesised and packaged by cells.

As such this work integrates very well with our other UKRI-BBSRC funded work leading to synergies and economies of scale.

While much of the machinery required for secretory cargo export has been defined from yeast genetics and in vitro reconstitution, recent years have seen the characterization of further modulators of the system. In particular, the export of procollagen from the ER has received much attention both because of its medical importance and because of its unusually large size and shape. We hypothesise a key role for the Mia gene family in integrating organelle structure with function in the early secretory pathway. Specifically, our preliminary data show a key role for Mia gene products in maintaining ER and Golgi structure. Work to date has focussed on two members of the Mia gene family, notably Mia2 (also called cTAGE5) Mia3 (and more specifically one splice form termed TANGO1) and work has been almost exclusively performed in vitro. Here, we will explore the diversity of this gene family, which arises through alternative splicing, to better understand this fundamentally important biological process. We propose to explore this diversity through analysis of the entire Mia gene family (Mia, Mia2 (which includes splice forms termed cTAGE5/TALI), and Mia3/TANGO1) in both human cell cultures and in zebrafish. We will define their requirements in 2D and 3D culture and in vivo. We will explore the diversity of cargo that they traffic, and the direct role in cargo capture versus roles in overall system optimisation. Zebrafish provide a simpler system in that fewer splice forms are present and gives us the opportunity to derive physiological insight into developmental and wound-related collagen synthesis and deposition. We will use gene depletion and knockout technology with high resolution imaging, proteomics, and biochemistry to define the role of these proteins in the fundamental organization of the ER-Golgi interface in vertebrate cells.