Litaf, a novel driver of membrane protrusion pathways

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences


The behaviour of cells within a tissue is controlled by their response to the environment. Receptor molecules at the cell surface receive a large number of chemical and physical stimuli that transmit signals to the interior of the cell and control important processes such cell migration, metabolism, cell proliferation and differentiation. Anomalies in the transmission of such signals result in pathological states that derive in diseases like cancer, diabetes, muscular dystrophy and neurological degeneration.

Amongst the most important signals that cells receive are from circulating small proteins called growth factors. These bind to specialised receptors, that in response to growth factor engagement alter their pattern of interactions with many molecules inside the cell to generate a so-called 'mitogenic response'. This response encompasses many changes to the cell's behaviour (such as growth, division and migration). One of the best-studied examples of a growth factor receptor is the epidermal growth factor receptor (EGFR), which controls many aspects of cell growth. Increased expression of EGFR is linked to several types of cancer. In order to prevent overstimulation of the cell response to growth factors, there are mechanisms of regulation to ensure that these responses are not sustained endlessly, which would lead to uncontrolled cell division and proliferation. These mechanisms encompass the internalisation of the receptor upon stimulation, leading it from the cell surface to specialised membrane-enclosed compartments within the cell (called endosomes). From endosomes, receptors pass to another membrane-enclosed compartment called the lysosome, where they are ultimately destroyed. The pathway to the endosome is termed the endocytic pathway.

We discovered key elements of the cellular pathway that controls the uptake of EGFR and its movement through the endocytic pathway. These elements recognise the stimulated receptor and re-shape the local membrane around it so that the receptor can move from the cell surface to endosomes and then to lysosomes. The current application concerns a new protein element that we have discovered, called LITAF. Our preliminary data suggest that LITAF can alter the shape of membranes and thus aid the transport of EGFR through the endocytic pathway.

The endocytic pathway is also critical in protecting against viral and bacterial infections and to eliminate protein aggregates that otherwise accumulate inside cells and result in the neurodegeneration observed in Alzheimer's, Parkinson's and Huntington's diseases. In fact, LITAF (lipopolysaccharide-induced TNF factor) was first identified as a protein that the cell produces in high quantity as a response to exposure to bacterial toxins. It is likely that the function of LITAF is linked in some way to the function of the endocytic pathway in helping to generate immune responses, though we do not yet know what the link is. In addition, mutations within LITAF that occur in the population lead to a debilitating paralysis termed Charcot Marie Tooth disease, caused by loss of myelin sheaths from the peripheral nervous system. We hope that information we gain from this project will help us understand how the health of the myelin sheath is normally maintained

In summary, the knowledge gained by our investigations will bring new insights into the molecular basis of many diseases caused by mutations in proteins that control the endocytic pathway, and in the long-term will guide further efforts for pharmacological intervention.

Technical Summary

Routing of cell surface-derived receptors through the endocytic pathway is fundamental to how cells interact with their environment. Defects in endocytic receptor sorting are linked to many diseases, including cancer and neurodegeneration and, hence, a comprehensive understanding of the endocytic pathway is of crucial importance. This proposal focuses on the function of LITAF (lipopolysaccharide [LPS]-induced TNF factor), a ubiquitiously expressed small integral membrane protein that is involved in sorting mitogenic receptors to the multivesicular body (MVB), but whose molecular function is uncharacterised. This project will build on preliminary data showing that LITAF is required at a late stage of MVB formation, and thus may drive deformation of the endosomal membrane in order to generate intralumenal vesicles within the MVB. We will then test our central hypothesis that LITAF is a key element in driving membrane protrusion away from the cytoplasm. This hypothesis will be tested from a number of directions.

We will identify the mechanism by which LITAF is integrated into the membrane of the endoplasmic reticulum, since we believe the membrane topology of LITAF may be key to explaining its biological function. For these experiments we will in vitro translate LITAF in the presence of canine pancreatic microsomes. We will also examine the interaction of recombinant LITAF with artificial lipid membranes. We will then test key structural determinants of LITAF, namely a putative Zn-finger domain, to test whether this plays a role in LITAF function.

Having identified the mechanisms by which LITAF integrates with lipid bilayers, we will examine whether this mode of insertion plays a crucial role for LITAF function at the MVB. We will also test more broadly whether LITAF plays a role in membrane protrusion processes, and to what extent these processes utilise the same pathways as MVB formation.

Planned Impact

Although the precise cellular function of LITAF is poorly characterised, it has emerged as a protein of great potential importance for both the biomedical and agricultural sectors. Hence, defining the cellular functions of LITAF will provide the essential groundwork for future studies into the underlying mechanisms of diseases as widespread as demyelination (Charcot Marie Tooth disease), cardiac disease, cancer and inflammatory disease (Crohn's disease). It is quite feasible that LITAF might offer new targets for novel therapeutic strategies.

The exposure of commercially important fisheries and livestock species to a range of pathogenic bacteria and viruses results in a many-fold induction of the LITAF gene. Hence, it has the potential to act as a biomarker for bacterial infection in commercial stocks, particularly when its physiological functions are clearly defined. This area of potential exploitation meets the BBSRC strategic priority of healthy and safe food.

The immediate academic impact of this project will be with research scientists working in related areas of the life sciences, including signalling receptor trafficking and cell migration. Our work will provide new knowledge, insight and scientific advances that will be relevant to all those that are active in these areas, irrespective of whether their research is being carried out from a basic or a medical perspective. Clearly, however, our work will direct relevance to clinical scientists. Another major stakeholder with a fundamental interest in this field is the pharmaceutical industry, with LITAF offering a potentially new clinical target.

The applicants are both strongly committed to engaging with the general public about the importance of Science and the specific goals of their research, actively participating in a variety of outreach activities. These range from Faculty and Institute open days to giving scientific presentations at English Literature conferences and informal spoken word events designed for a wide audience. We strongly encourage our staff and students to take part in raising the profile and understanding of science in the wider society, and Ling Zhang the PDRA will participate in outreach activities throughout the course of the project. In addition, the project offers further scope for Ling to develop his range of advanced EM skills, making him well placed to pursue a long-term career in science, thereby contributing to the UK knowledge base and economy. Since Ling is a Chinese national, there is also an important international context to his academic development.


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Description We have examined how a protein called LITAF (LPS-induced TNF factor) inserts into biological membranes. LITAF is an important protein, since mutations in LITAF give rise to Charcot Marie Tooth disease, a demyelinating neuropathy. Hence, understanding precisely how LITAF works, and what it does within cells, is important for understanding the pathology of this disease. We have found that LITAF inserts into biological membranes in an unusual way, which may be important for its biological function. We have attempted to establish the biological function of LITAF. We find that LITAF is associated with endosomes, and that loss of LITAF affects the endosomal recycling of integrins. Hence, LITAF appears to be involved in maintaining the complement of cell surface proteins, which may be the root cause of Charcot Marie Tooth disease in patients carrying LITAF disease mutations.
Exploitation Route We published the findings in an academic journal, so that they can be utilised by other scientists and by clinicians.
Sectors Other

Description Adam Reid 
Organisation University of Manchester
Department School of Earth and Environmental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution The joint project is to examine the impact of LITAF on Schwann cell function and on the repair functions of Schwann cells. Our laboratory provides reagents, ideas and expertise in endosomal trafficking.
Collaborator Contribution The Reid laboratory provides reagents, ideas and expertise in Schwann cell biology.
Impact We have submitted a joint grant proposal to Muscular Dystrophy UK and another grant proposal to MRC
Start Year 2016
Description Janni Petersen 
Organisation Flinders University
Country Australia 
Sector Academic/University 
PI Contribution We are examining the relationship between endosomal trafficking and TOR nutrient signalling. We provide expertise and reagents in endosomal trafficking.
Collaborator Contribution We are examining the relationship between endosomal trafficking and TOR nutrient signalling. Dr Petersen provides expertise and reagents in TOR signalling.
Impact We are currently writing joint grant applications.
Start Year 2016
Description Stephen High LITAF 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We are exploring the function of a novel membrane protein called LITAF (LPS induced TNF activation factor).
Collaborator Contribution Stephen High examines membrane protein topology. Using High's expertise, we have established the membrane topology of LITAF. This is a new contribution to the field of membrane trafficking.
Impact No outcomes so far
Start Year 2014
Description BioDiscovery Days at Manchester Museum 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact BioDiscovery Days at Manchester Museum was organised for March 2020 as part of British Science Week. To celebrate British Science Week the School of Biological Sciences showcased the BioSciences, with BioDiscovery Days on Thursday 12th and Friday 13th March 2020, hosted by Manchester Museum. The event showcased the work of over 40 researchers from different faculties of the University. My lab joined the event.
The event was aimed at KS3 pupils (year 7-9) but is also suitable for year 10 pupils, to highlight some of our ground breaking research in the biological and biomedical sciences
Visitors attended the bioscience fair, where stands offering interactive activities showcased a wide range of different biological topics. There was the opportunity to participate in workshops and activities to enhance understanding of some of our current research in the biological sciences. These activities, in conjunction with lightning talks raised awareness of the different options available to study these subjects at Manchester (and elsewhere), and explain the kind of careers these subjects can lead on to.
There were also opportunities for school students to meet and interact with some of University of Manchester undergraduate and postgraduate students, as well as our research and teaching staff.
During each day, there was a session (10am to 12.30pm) where researchers presented to invited secondary school groups. Students were encouraged to visit research stations. In addition there were workshops, lightning talks, film reels and storytelling.
My lab (including post-docs and students funded by the BBSRC) generated a research station centred around sub cellular organelles; what they look like and what they do. The research station allowed us all to get chatting to students about science, both specifically about how cells work and in general.
Year(s) Of Engagement Activity 2020