Organelle dynamics and function in the late endocytic pathway

Cells are compartmentalized by specialized organelles (little organs within each cell), and cargo including proteins is moved between these compartments by the trafficking of vesicles (little bubbles surrounded by a thin membrane made of a fatty substance called phospholipid and studded with proteins). Some of the cargo is taken up into the cells by vesicles formed at the cell surface that deliver their cargo to organelles called endosomes, endolysosomes and lysosomes. Lysosomes act as storage organelles for enzymes that degrade cargo that is delivered from the cell surface or other parts of the cell. Degradation occurs in endolysosomes formed by the fusion of endosomes with lysosomes. Lysosomes are regenerated from endolysosomes by a process that is currently poorly understood and which we will study. These lysosomes can then be re-used. We aim to identify and study the proteins that make up the machinery necessary to achieve and regulate the regeneration of lysosomes. Our work will help us to understand much better why the organelles we are studying are so dynamic and to explain how the environment within them is regulated such that endolysosomes are such an efficient site for cargo degradation and lysosomes work so well to store the degradative enzymes. There are many diseases in which defects in the cell surface and/or in membrane traffic to lysosomes occur. These include rare genetic diseases such as the lysosomal storage disorders and more common diseases including diabetes, atherosclerosis and neurodegenerative diseases as well as infectious diseases where microbes subvert the membrane traffic system in order to infect cells. In the long term our work will contribute to developing better ways of targeting drugs to particular sites within cells for more specific drug therapies.

We will study organelle dynamics in the late endocytic pathway of mammalian cells, focussing on the reformation of lysosomes from endolysosomes. Endolysosomes, formed by endosome-lysosome fusion, are acidic, acid-hydrolase-active organelles from which neutral, acid-hydrolase-inactive, re-usable lysosomes are reformed. We will use small molecule inhibitors, rapid knocksideways inactivation and subcellular fractionation/proteomics approaches to identify protein machinery required for lysosome reformation from endolysosomes. We will investigate the role of the V-ATPase (vacuolar H+ ATPase) in the regulation of acidification during lysosome reformation and lysosome-endosome fusion and, using expressed fluorescent protein-tagged subunits, we will test whether regulation of lysosomal V-ATPase is regulated by the reversible dissociation of VO and V1 sectors, removal of the whole complex and/or binding of other proteins. We shall compare the machinery of lysosome reformation from endolysosomes with that used in lysosome reformation from autolysosomes and we will probe the relationship of intracellular positioning of endolysosomes/lysosomes to their state of maturation in the lysosome fusion and regeneration cycle. Throughout our study we will use confocal fluorescence microscopy, live cell microscopy, super-resolution microscopy, immunoEM and CLEM (correlative light and electron microscopy) of cultured cells to study lysosome reformation and as we uncover protein machinery involved in lysosome reformation and V-ATPase regulation we will use an integrated structural and cell biology approach to investigate protein interactions.

In addition to the specific academic beneficiaries that have been listed in the academic beneficiaries section above, the pharmaceutical industry, the general public and the wider academic and clinical community will benefit from our greater understanding of the basic cell biology organelle dynamics and function in the late endocytic pathway including our understanding of the functions of late endosomes, endolysosomes and lysosomes. Benefits will occur as follows:
1. Benefits for Industry: The basic research outlined in this proposal is likely to be important to the pharmaceutical industry in two specific ways:
(i) There is currently renewed interest by the pharmaceutical industry, in late endocytic organelles, because of the accumulation of weakly basic amphiphilic drugs in such organelles with consequences for drug pharmacokinetics, drug-drug interactions and off-target toxicity events. Paul Luzio and Nick Bright will continue to have meetings with scientists in the pharmaceutical industry working in biotransformation, drug disposition and drug safety to share new information about the properties and functions of endolysosomes/lysosomes and to discuss ways of assessing lysosomal 'health' during drug development programmes;
(ii) Identifying and characterizing the molecular machinery driving the dynamics of the late endocytic pathway has the longer term prospect of identifying targets for therapy both in lysosomal storage diseases where a complex cell pathology develops as a result of the 'jams' occurring in membrane traffic as a consequence of undigested material build-ups in endolysosomes/lysosomes and infectious diseases where microbes hijack the late endocytic pathway. Such targets are likely to be of interest to those sections of biotec and pharmaceutical industry interested in novel approaches to treating infectious diseases. To explore potential technology transfer we will seek advice and use expertise from Cambridge Enterprise, Cambridge University's technology transfer office.
2. Benefits for the General Public: The general public will benefit from this research, because it will help our understanding of the causes of diseases resulting in abnormal function of the organelles and traffic machinery of the late endocytic pathway. Explaining why these organelles are important for health, what can go wrong and how repair may eventually be achieved will have an educational impact. We will use presentations at the Cambridge Science Festival as well as on our Institute website and the CELLpics website for schools (http://cellpics.cimr.cam.ac.uk/), as a way to get our new findings across to the general public including patients with defects in the late endocytic pathway.
3. Benefits for the wider academic and clinical community:In the long term health care professionals, both academic and clinical as well as patients will benefit from the work undertaken in this study especially if new knowledge about organelle dynamics in the late endocytic pathway can lead to better drugs that do not accumulate in the acidic environment of these organelles and if new targets for therapy are discovered. In the long term there may be impact on the well-being and quality of life of patients as well as economic benefits by saving health care costs and by initiating activities in the UK-based pharmaceutical industry.