High-resolution imaging and time-resolved proteomic profiling of COPII-dependent procollagen packaging.

Collagen is the most abundant protein in our bodies. It is also one of the largest. This size presents a significant problem to cellular machinery involved in shipping procollagen from its site of synthesis (inside the endoplasmic reticulum of cells) to its site of action in the extracellular matrix. The machinery that controls the first step in this pathway normally generates transport carriers that are simply too small to accommodate procollagen.

Nearly all cell types secrete one or more types of collagen and this process is absolutely essential during development to provide the extracellular matrix within which cells migrate to their final position and differentiate. It is also essential for those very differentiation processes. Later in life ongoing secretion of collagen is vital for tissues to maintain themselves by renewal and regeneration. Major pathologies are also associated with defects in collagen secretion including fibrosis. The field of stem cell biology increasingly relies on accurate reproduction of the "stem cell niche", of which the cohort of secreted collagens is a major component. The development and exploitation of engineered cells and tissue for therapeutic benefit will in many cases rely on a complete understanding of these processes. Consequently it is perhaps surprising how little we know about this process. The machinery that directs the first step in the secretion of small proteins (for which a 2013 Nobel Prize was awarded) has been very well characterised.

This machinery generates small (60-80 nanometre) transport vesicles that are not sufficient to encapsulate the ~300 nanometre size of collagen molecules. While there have been some advances in this area, they either relate to specific collagen types (such as collagen VII) or are underdeveloped in terms of our understanding (such as modification of the core machinery using a small protein called ubiquitin). Our work aims to use a combination of cutting-edge imaging and proteomics methods to precisely define this machinery, and the nature of these transport carriers exactly at the point of procollagen export. Importantly we will focus on two major procollagen isotypes - I and II to provide insight into the pathway for these forms that are of major clinical interest. We have generated new tools to examine this process with unprecedented spatial and temporal precision. As such we anticipate making major advances in this important field.

The mechanisms of secretion of the most abundant protein in animals remain unclear. While much is known about the core machinery that drives export of procollagen from the ER, and several high profile papers have defined additional components, we still simply do not know how the major collagens are exported from the ER. The majority of the available mechanistic data either relates specifically to one procollagen isotype (type VII), or provides little insight beyond the initial finding (in the case of ubiquitylation of Sec31), or relate to the downstream processes of tethering and fusion. We have developed some unique methodologies to define this step for the most abundant and important collagens, types I and II. We have generated cells to express engineered constructs to enable temporally- and spatially-resolved imaging as well as proteomics of procollagen I during exit from the ER. The key advance here is the use of a biotin- and ascorbate-dependent procollagen construct to control export from the ER, imaging using a monomeric GFP. Imaging experiments will use correlative light electron microscopy, exploiting the unique expertise and previous RCUK investment in these areas within Bristol. Complementary methods will exploit the proximity-based labelling method of engineered ascorbate peroxidase (APEX2) to define those proteins within close proximity to procollagen on both sides of the ER membrane during its export. Perhaps most significantly, we have shown that the biotin-phenol substrate used for APEX2 will, like biotin itself, drive export of tagged constructs from the ER. This means we can combine these approaches as we seek to correlate super-resolution light microscopy, higher resolution electron microscopy, and proteomic profiling.

These experiments will provide a major advance in our understanding of this essential process that underpins key aspects of development, wound repair, fibrosis, and cancer biology.

There are key aspects within the project that have potential to be of use in the pharmaceutical and related industries. There is great interest in the possibility to subvert existing cellular pathways for therapeutic benefit. In addition, the dysfunction of these pathways is either a direct or underlying feature of many human diseases. In recent years, several human congenital diseases have been determined to be caused by mutations in genes encoding membrane trafficking machinery. These diseases span a range of physiological steps from skeletal development to neuronal function. This highlights the importance of a full understanding of these pathways to guide possible future clinical intervention. Thus, the potential impact of our work is without question. While it is always more complex to define the way in which and timescales for such impacts might occur, we can develop such lines through our impact plan. Through informing our basic understanding of a critical cellular process, it is most likely our work will inform long term projects in other fields including the pharmaceutical industry.
In this way, potential applications of this work are identified from within the department (through regular discussion with our Impact lead and industrial liaisons) as well as by continuing discussions with our Research and Enterprise Department. Any outcomes of this work that are exploitable, notably in terms of intellectual property or knowledge transfer to the private sector, are handled by the highly experienced team within RED; who engage closely with funders when appropriate. As with all of our projects, this one includes considerable opportunity to train the researchers involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration with public communication and outreach programmes and the extensive network of University schemes to benefit the training and development of research staff (Bristol is at the forefront of research staff development). I have a good track record in facilitating the placement of staff in areas outside our core research activity. For example, a previous postdoc in the lab undertook a period of flexible working in order to shadow some of our Research and Enterprise team and subsequently undertook a part-time course in intellectual property management. She has now moved to such a position with a major company working in this area. This demonstrates that the environment provided by my own lab a well as the University as a whole is highly conducive to career development of our staff beyond academic, basic science research alone and thus contributes to the economic development of the nation. Our projects are also very data intensive - notably from imaging work - and the management and analysis of such large (terabyte) datasets is applicable to many areas of professional life.
This work will lead to significant image data that is readily used in both public understanding of a science and artistic arenas. Examples include local exhibitions and promotions. Through our public engagement plans, entering competitions, and other outreach activities, this work therefore is likely to contribute to local exhibitions or displays as has been the case with previous work from our lab and others within our School.