The role of OCRL1 in endocytic membrane traffic

The cells in our body are made up of internal membrane-enclosed compartments called organelles, each with their own identity and specific functions. Material is transferred between the organelles and between certain organelles and the outside of the cell via a process termed membrane traffic. One of the major membrane traffic pathways is the endocytic pathway, where material is taken up from the cell exterior via engulfment in membrane-enclosed vesicles at the cell surface. The internalised material is then sorted to various downstream compartments or recycled back to the cell surface. The endocytic pathway is fundamentally important to all cells, and when defective gives rise to numerous diseases in humans. Moreover, this pathway is exploited by pathogenic viruses and bacteria to gain entry into cells to cause infection. It is therefore important we understand how the endocytic pathway functions at the molecular level.

This proposal is focussed on two enzymes that are localised to organelles within the endocytic pathway, called OCRL1 and INPP5B. These enzymes act on a class of lipids called phosphoinositides that are important for controlling various aspects of cell behaviour including endocytic membrane traffic. Mutation of OCRL1 causes two diseases in humans, called Lowe syndrome and Dent-2 disease, which are characterised by defective brain, eye and kidney function. The mechanisms by which mutation of OCRL1 brings about the pathological changes seen in Lowe and Dent patients remain unclear. We have identified a new binding partner for OCRL1 and INPP5B called IPIP27A that is essential for trafficking within the endocytic pathway. The aim of this proposal is now to determine how IPIP27A functions together with its interaction partners to regulate endocytic trafficking. Our hypothesis is that OCRL1/INPP5B and IPIP27A participate in the formation of membrane carriers on endocytic organelles to ensure trafficking occurs in a properly controlled manner. How this process occurs remains poorly understood. We plan to use a combination of experimental approaches to dissect the mechanisms involved. This will entail analysis of endocytic trafficking in tissue culture cells using various established methods including imaging of living cells in real time.

Our work will provide important knowledge as to how the endocytic pathway functions in all cells, as well as reveal the mechanisms underlying Lowe syndrome, Dent disease and other disorders caused by defects in endocytic trafficking.

Phosphoinositide (PI) lipids are vitally important for a multitude of cellular processes ranging from intracellular signaling to membrane traffic. The abundance and distribution of PIs is under tight control by kinase and phosphatase enzymes that consequently are important for normal cellular and organismal physiology. One such enzyme is OCRL1, a PI 5-phosphatase whose mutation causes two disorders in humans, Lowe syndrome and Dent-2 disease. OCRL1 has a role within the endocytic pathway, where it regulates receptor recycling, but how it carries out this function is poorly understood and consequently the mechanisms underlying Lowe syndrome and Dent disease are ill defined. Endocytosis is a universally important trafficking pathway present in all eukaryotes. Its dysregulation results in disease, and it is often exploited by bacterial and viral pathogens to cause infection. We have recently identified IPIP27A as a novel OCRL1 interactor with a key role in endocytic recycling. We have gone on to identify the cellular machinery to which IPIP27A binds, which includes several trafficking components associated with the actin cytoskeleton. These findings strongly support a model whereby OCRL1, IPIP27A and its associated proteins regulate the formation of carriers that mediate endocytic recycling. The aim of this proposal is to test this model and define the mechanisms by which OCRL1, IPIP27A and its actin-associated interactors function in carrier formation. To achieve this aim, a variety of cell-based approaches will be used, including trafficking assays, live cell microscopy and electron microscopy. Together our studies will provide significant insight into how membrane traffic within the endocytic pathway is regulated by PIs, and how defects in this process cause disease in humans.

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

Beneficiaries of the research
We envisage that findings of our current study will be of interest to the public at large given the clinical importance of the proteins we will investigate in the proposal. We will communicate our findings to the public via the open access University website and through the Faculty Research brochure. We will also notify the dedicated Faculty press officer of our findings at around the point of publication in academic journals. The press officer will then contact local and national news agencies, and if appropriate prepare press releases that these agencies can use. The Lowe laboratory has participated in several public engagement initiatives and we plan for this to continue. The lab has hosted two A-level students at different times as part of the Nuffield bursary scheme. The Faculty of Life Sciences continues to participate in the Nuffield scheme and we envisage hosting another A-level student should the current application be successful. Members of the Lowe lab have participated in several public engagement exercises involving visiting schoolchildren, hosted at the Manchester Museum with whom our Faculty has close links. Members of the Lowe lab will continue to participate in these exercises in the future.

ML has been involved closely with the Lowe Syndrome Trust, a registered UK Charity dedicated to supporting Lowe syndrome patients and their families. This has included providing scientific advice, helping in the lay understanding of scientific discoveries relevant to the disease, and in supporting public functions arranged by the Charity. The current application is highly relevant to this disease and findings arising from the work will be of significant interest to the Charity. This will be communicated to the Charity through personal contact. We will also continue to support the fundraising and public engagement work of the Charity.

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 of human disease mechanisms that in turn will aid the design of better treatments for patients. This is particularly relevant to Lowe syndrome and Dent disease, but more broadly will inform research into diseases such as Alzheimer's, for which the IPIP27 partner CD2AP is a known risk factor, glomerular disease, caused by loss of function of CD2AP and another IPIP27 interactor myosin 1E, and diseases associated with dysfunctional endocytic recycling.

The technical and intellectual knowledge acquired by the post-doctoral researchers during the course of the proposed work will equip these people to pursue a career in science or science-related discipline. The researchers 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.