Novel zebrafish approaches to investigate leaderless protein secretion in vivo

All cells within the body traffic their proteins either to compartments within the cell, or to the cell surface. At the cell surface they can either remain attached to the cell or they are secreted so that these proteins can be "seen" outside the cell, and away from the cell. The vast majority of surface or secreted proteins have a specific signal directing them to automatically traffic there. Recently it has been found that a small number of proteins, which play a role outside the cell but were assumed to be externalised only upon cell death, are in fact secreted under certain circumstances. One group of secreted proteins are inflammatory cytokines (messengers), critical in signaling to blood cells the location of an injury or infection and so essential to maintain a healthy immune system throughout life.

We now know that activation of the damage-sensing "P2X7" cell surface receptor causes release (secretion) of inflammatory mediators (specifically "interleukin-1) into the bloodstream. Interestingly, when interleukin-1 exits the cell, it is enveloped in small, protected membrane packets, that we have termed "microvesicles". These packets are likely to prevent this highly inflammatory cytokine from acting at random sites within the body, and may provide a means of targeting it to particular cells where the microvesicles can dock.

The cellular machinery coordinating microvesicle formation and cytokine secretion is complex. Several pathways involved in this process have been proposed, based on studies using a single type of cell grown in culture. We have developed a new system where we can study fluorescent cytokine in a whole live organism, namely the transparent zebrafish larva. For the first time, this offers us the opportunity to view the cells that secrete interleukin-1, and to see where interleukin-1 is targeted in a live organism. In this project we will optimise computational approaches to quantify vesicle formation, tracking and targeting in a whole organism using our fluorescent zebrafish lines. We will then test which cellular components are responsible for interleukin-1 secretion by adding inhibitors to these components and measuring changes in response to an infection. We will also use these fluorescent zebrafish to screen for small molecules that are effective in blocking inflammation, so that we can identify the critical parts of the cell that are required to for a healthy immune response. In the future, beyond this project, this will help us to develop new therapies to fight disease.

Secretion of leaderless proteins is important in a range of contexts, but poorly understood. Specific proteins have been identified lacking a leader sequence and secreted independently of the conventional ER-Golgi route including fibroblast growth factors, interleukins and galectins. The importance of this unconventional secretion pathway is underlined by the tight regulation seen in the secretion of the best-studied example, the pro-inflammatory cytokine, interleukin-1 (IL-1).

Whole animal models have been used to evaluate the requirement of specific proteins in permitting leaderless protein secretion whilst cell culture studies have allowed us to observe whether these pathways are vesicular or otherwise. It has not so far been possible to combine the key features of such models to determine, in an intact live organism, whether leaderless protein secretion is vesicular and how this enables IL-1 targeting. We have recently characterised the zebrafish as a model of vesicular IL-1 secretion and targeting in response to injury and pathogens, and have generated specific transgenic lines that permit novel studies of leaderless protein secretion mechanisms in vivo.

Our work has led us to hypothesise that the key to controlling IL-1 function is the regulation of vesicular release of IL-1 via components of the vesicular budding pathway and disruption of cell membrane lipid diffusion. We will use our zebrafish lines to establish this as a widely applicable model to study IL-1 processing, leaderless protein secretion more generally, and to identify the in vivo extracellular targeting of these proteins. We will generate rapid, high content quantitative assays of IL-1 function in vivo for application to downstream objectives & investigate whether components of the vesicle budding pathway or of membrane lipid diffusion are required for the release of leaderless proteins in vivo, with important consequences for the regulation of fundamental processes such as inflammation.

The proposal addresses fundamental cell biology questions of how leaderless proteins are secreted from the cell. There are less than 40 known leaderless proteins within the cell, a significant proportion of these play a critical role in inflammatory responses to injury or infection. The discoveries from this project will enhance the knowledge economy with new scientific advancement, as described in 'academic beneficiaries".

There is considerable interest from the pharmaceutical industry in discovering regulators of leaderless protein secretion which are fundamental in inflammation and become dysregulated and chronic in a number of ageing diseases. We anticipate that the unique nature of these secretion mechanisms will make them attractive targets for anti-inflammatory therapeutics. Protection of IP for these targets as they are discovered will bring significant economic gains to UK plc. Integration with the Pharma industry will allow rapid drug development, building on existing collaborations such as Renshaw's MRC Industry Partnership Award with GSK.

The project will establish and validate assays for the future identification of new small molecular inhibitors of leaderless protein secretion and/or targeting in inflammation. This leads to obvious longer-term commercial opportunities to develop the technology to a higher throughput level, and to engage industrial partners in developing new therapies. Via existing and new links, we will encourage Pharma investment in this programme, and develop IP sharing arrangements to ensure mutual benefit from emerging knowledge and know how. The advances in knowledge, and potential for driving drug development will ultimately impact on quality of life, health and well-being. Avoidance of dysregulated inflammation is a prerequisite for healthy ageing.

The project uses cross-disciplinary approaches from mammalian cell biology, zebrafish models and systems engineering. These approaches will be used to develop and make use of innovative systems technologies to identify, track and quantify accumulation of secreted leaderless proteins, in vivo. The project will contribute to new expertise in developing these unique tools to address biological questions by a systematic, and ultimately, high throughput approach. The project will strengthen links between these different disciplines and forge a greater understanding of how we can engage, complement and enhance research for the future.
This proposal will deliver highly trained researchers offering unique skills. The researcher will combine skills in fish models and state of the art in vivo microscopy, in parallel with mammalian cell based assays. The researcher will develop distinctive skills in generating new computational software to provide a systems approach to understanding biological responses. This expertise will provide transferable skills to other non-academic beneficiaries, but will also be used to train researchers from other groups in our methodologies. The new software developed will be made freely available to academic community, but may contain novel elements, which will provide additional opportunities in knowledge transfer and commercialisation.

The co-investigators are actively involved in public engagement and broader dissemination, with regular school visits and high level involvement with public exhibitions such as Royal Society Summer Science exhibition, and the University of Sheffield Festival of the Mind.