Defining the architecture of the endosome-specific ESCRT-I complex

The behaviour of cells within a tissue is controlled by their environment. Amongst the most important signals that cells receive are from circulating small proteins called growth factors. These bind to specific proteins, called receptors, which are found on the surface of cells. Binding of growth factors alters the shape of receptors (the receptor is turned 'on'), and this in turn changes the pattern of interactions between receptors and many molecules inside the cell that control cell growth and division. In this way growth factor receptors act as essential bridges between the cell exterior and interior to stimulate so-called mitogenic, or growth, responses. In order to prevent these responses continuing endlessly, which would lead to uncontrolled cell division, the growth factor receptor must be sent to an environment where it can be permanently turned 'off'. Ultimately, it is sent to a specialised compartment within the cell, called the lysosome, where it is destroyed.

Movement of the receptor from the cell surface to the lysosome involves the receptor being sequestered into regions of the cell surface membrane that invaginate and pinch off to form spherical packages, or vesicles, within the cell interior. These vesicles first move to and coalesce with an intermediate compartment called the endosome, which is rather like a balloon. Importantly, the growth factor receptors are still active when they reach the endosome. To ensure they are stopped from working, they are enclosed within little vesicles that are forced within the inside of the endosome. This occurs by a process of inward budding, rather like poking deep impressions into a balloon and imagining these could pinch off to form internal packets. This process means that the mitogenic receptors are now completely separated away from the rest of the cell contents and unable to work. The endosome, along with these internal packages, is then sent to the lysosome.

The aim of this project is to understand how activated mitogenic receptors, once they reach the endosome, are packaged into the interior of the compartment. The project will focus on examining the composition and shape of a group of proteins which assemble into a 'protein complex' that take part in this event. It is crucial, in order to understand how this protein complex works, to find out how it is assembled and which parts of the complex are able to reach out and grab the receptors, as well as those parts that bring additional important proteins and protein complexes to the site of receptor packaging.

There is one additional important reason for providing accurate information about how this protein complex is assembled. Like many cellular proteins or protein complexes, several of its structural features are shared by proteins/protein complexes that are involved in completely different cellular activities. Therefore, providing very accurate information about what this protein complex looks like will help generate tools that can interfere with this and only this protein complex. This should be a real advantage when it comes to designing research tools or clinical tools that selectively modulate the activity of this complex and therefore influence mitogenic receptor lifetime but not other important cellular processes.

Activated tyrosine kinase signalling receptors such as epidermal growth factor receptor (EGFR) are endocytosed, ubiquitinated, and then sorted to intralumenal vesicles (ILVs) within the multivesicular body (MVB). MVB sorting is essential for EGFR down-regulation, and occurs via a series of ESCRT (Endosomal Sorting Complex Required for Transport) complexes (ESCRTs 0-III). These, collectively, recognise ubiquitinated cargo and deform the endosomal membrane to generate ILVs.

ESCRT pathways are now known to facilitate many very different cellular functions besides MVB sorting, so a crucial question is how each of these pathways can operate independently. One hypothesis is that each cell function requires a unique set of ESCRT complexes, defined by specific subunits that have evolved to work optimally in each context.

We have recently shown that this is the case for ESCRT-I, the key complex involved in orchestrating multiple ESCRT pathways. The ESCRT-I complex involved in MVB sorting has unique features compared to other ESCRT-I complexes, including multiple ubiquitin-binding domains and an unusually large hydrodynamic radius. These properties are imparted by specific subunits, VPS37A and UBAP1.

It is now essential to fully define the architecture of this endosomal ESCRT-I. First, by dissecting how VPS37A and UBAP1 recognise each other and assemble into the ESCRT-I core, these studies will help us understand how functionally diverse ESCRT-I complexes can self-assemble. This information has wide-ranging implications for a range of cell biology fields. Second, the studies will provide a comprehensive picture of the global architecture of the complex, including the juxtaposition of functionally important domains. This information will drive the construction of working models for how this ESCRT-I complex can integrate recognition of ubiquitinated EGFR with activation of the downstream ESCRT pathway.

The work proposed in this project addresses important questions in basic scientific research that will interest the wider scientific community. However, as the target of our study is a protein complex that includes two tumour suppressors and a validated risk factor for neurodegenerative diseases (Frontotemporal lobar dementia; FTLD), the impact of the research will be of interest to the general public, the medical and clinical community and the pharmaceutical industry. The primary means of informing the latter communities of our work is through publication in the scientific literature. The PIs and RAs will all be expected to play an active role in disseminating information to increase the impact of the research here. They will present their work at both national and international conferences.

Both cancer and neurodegenerative diseases inflict a very high burden on the health system and society in general, as they require long-term specialised care and expensive treatments. This impacts negatively on the global economy as these diseases target both working age population as well as the elderly. Any contribution towards the understanding of the basic biological processes that underlie such pathologies will have a long-term, yet significant, economic and strategic benefit for the UK and will result in a higher quality of life worldwide.

A detailed understanding of the structure and function of our target protein complex at the molecular level may serve as the basis for the design of modulators of its function which could either perturb or promote interactions with biological partners. In the long-term, these strategies could be applied to control defects in pathways leading to cancer, such as EGFR signalling events where our target is implicated, and to treat hereditary diseases associated with mutations in endosomal effectors.

We will communicate our findings to the public via the University website and through the Faculty Research brochure. We also have a very active Faculty press office that ensures important scientific findings are publicly advertised in the local and national press. Dr. Tabernero has worked closely with that office in the past to arrange the publication of her research in the Manchester Evening news and subsequently was interviewed by the BBC and appeared in a documentary on tuberculosis. Her work has been the focus of several public engagement activities and disseminated to the general public in open lunch seminars at Nowgen (NIHR), The Royal Society of Medicine (London) and the Health Protection Agency meetings. We also plan to engage young budding scientists by contributing to the seminar talks organised by the Philosophical Society at various leading schools in Manchester. Prof. Woodman has close links to the Manchester Museum where his lab has participated in a number of public engagement activities to disseminate his research (Science days at the Museum, Science Fairs in Manchester).

One of the primary outcomes will be specialist skills training for the named RAs, Deepanker Gahloth and Kim Brownhill. Whilst both have a range of expertises that they bring to this project, they will be working in an environment that is conducive to further technical training. In particular, the biophysical analysis will take place within the FLS Biomolecules Facility, and both Kim and Deepanker will receive training from Dr Tom Jowitt, who runs the facility. We have worked closely with Tom over several years. His work towards training post-docs in our laboratory has so far been recognised by his authorship on a high impact paper, with a further paper in preparation. The training that Deepanker receives is particularly important in a global context, since Deepanker is an Indian national who has come to work in Manchester. Hence, the work will make an important contribution to the international mobility of scientists.