Mechanism and function of organization of secretory cargo export from the endoplasmic reticulum.

The internal organization of mammalian cells has been well documented since the dawn of cellular microscopy. It is therefore quite astonishing how little we know about why the cell is laid out in this way - in simple terms, how does form dictate function? A great example of this comes from the organization of the secretory pathway. All proteins secreted from your cells go through this pathway - all hormones (insulin, growth factors, digestive enzymes), all neurotransmitters, all antibodies made by your immune cells. Furthermore, all of the proteins that sit on the surface of your cells to recognize these key signalling molecules are put there by the secretory pathway. Indeed, all internal cell membranes are composed of proteins that are targeted to these individual compartments by the secretory pathway and serve to define the identity of the membranes where they reside. Why then is the first step of the secretory pathway organized at discrete sites known as transitional ER (tER). These spots that can be seen in the cell when labelled with specific markers are the first site of spatial organization of the secretory pathway. As such, in the same way that the foundations define the architecture of a building, these sites define the architecture of the cell. We and our new collaborators have identified two of the proteins that define these tER sites and now wish to go on to show how these act together to generate these sites and how their interdependent function relates to the core organization of the tER. Following on from some of our preliminary data, we are now in a position to ask the fundamental question of how tER organization directs function. We have evidence that we can disrupt organization without blocking core activity. We propose to investigate the role of this organization in the core activities of the secretory pathway and in particular focus on the role of tER organization in the secretion of the most abundant protein in your body, collagen. We have good evidence that these core proteins play a key role here and some evidence that they might even be involved directly in processing collagen during its synthesis. Funding this project will enable us to make major contributions to our fundamental understanding of cellular organization and function. Such findings have good potential for relevance to those seeking to study the dysfunction of this pathway in disease as well as those seeking to manipulate it for therapeutic benefit (the production of clinically active secreted molecules, modifying the secretory load of cells etc).

This proposal seeks to define the molecular basis of the organization of the exit point for secretory cargo from the endoplasmic reticulum. We have identified two key proteins that interdependently function in this step and propose to define their relationship using a combination of molecular cell biology and in vitro approaches. Our background in imaging underpins both sides of this proposal and we have engaged key collaborators to advance those aspects with which we are less familiar. COPII-dependent trafficking of secretory cargo from the ER to the Golgi represents the first membrane trafficking step in eukaryotic cells. In metazoan, this event is organized spatially at discrete sites on the endoplasmic reticulum (ER) membrane called transitional ER (tER). In recent years, we showed that these sites are defines in mammalian cells by Sec16. This protein serves as an organizer defining the site of assembly or other COPII proteins during vesicle formation. The lab of Jon Audhya has now identified TFG as a direct mediator of Sec16 clustering at tER sites. Audhya's initial work from C elegans leaves many questions as to the role of TFG - whether at the tER itself of further downstream in directed movement of vesicles. We propose to define the interdependent roles of TFG and Sec16 in mammalian cells - our hypothesis is that they act together to define the tER both functionally but also structurally. We propose to reconstitute this in vitro to determine the molecular basis for tER site assembly. We also have evidence that these proteins act in concert to direct collagen secretion and we will explore this avenue using a series of well established assays. Together these experiments will define the molecular basis for the organization of the transitional ER and to address why this organization relates to function.

There are key aspects within the project that have potential to be of use in the development of technologies within 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 the ER export machinery. These diseases span a range of physiological steps from skeletal development to erythropoiesis. This highlights the importance of a full understanding of these pathways to guide possible future clinical intervention. It is also anticipated that other diseases such as cystic fibrosis might be ameliorated by enhancing the efficiency of ER export and indeed clinical efforts along these lines are well developed. 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. For example, should we define that the organization of the secretory pathway is central to the secretion of extracellular matrix, it is most likely that this work will impact on the field of regenerative medicine. At this point we would engage with those contacts we have in this area to explore possible areas of therapeutic benefit (e.g. the group of Anthony Hollander in Bristol). In this way, potential applications of this work are identified from within the department as well as by continuing liaison 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 such as MRC when appropriate.

As with all of our projects, this one includes considerable opportunity to train the researcher involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration without public communication and outreach programmes, 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 (MRC-funded) 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.