MRC DiMeN Doctoral Training Partnership: Vaulting the void: The vault particle as a novel mediator of intercellular communication in the tumour microenvironment

Background

The vault particle is the largest known eukaryotic ribonucleoprotein complex. It is composed of 78 copies of major vault protein (MVP) as well as two associated proteins (TEP1 and PARP4) and vault RNA (vtRNA) [1,2].

The function of the vault particle remains elusive. However, it has been implicated in intracellular transport and multidrug resistance. VtRNA have been linked to resistance to apoptosis and chemoresistance, independent of the vault particle [3].

Cancer cells release a complex mixture of soluble factors and extracellular particles into the tumour microenvironment (TME), which have been implicated in driving tumorigenesis. In recent decades, extracellular vesicles (EVs) have attracted attention due to their role in intercellular communication in the TME. MVP and vtRNA have repeatedly been reported as EV cargo. However, a recent report [4] (and our unpublished data) demonstrated the presence of intact vault particles in the extracellular space. We have shown that vault particles are frequently co-isolated with EVs.

Experimental approach

In collaboration with our industrial partner, NanoFCM, we will utilise the state-of-the-art NanoAnalyzer to characterise the extracellular particles released from wild-type oral cancer cells and a vault particle deficient cell line (already generated in house). You will receive full training to use the NanoAnalyzer and will spend at least 3 months throughout the PhD working at the NanoFCM laboratory in Nottingham. You will also become proficient in cell culture, enrichment of extracellular particles (using techniques such as size exclusion chromatography and ultracentrifugation) and fluorescent labelling of extracellular particles.

We will investigate the mechanism of vault particle release using advanced microscopy techniques such as confocal microscopy and transmission electron microscopy coupled with immunogold labelling. We will use small interfering RNA and CRISPR Cas9 gene editing to perturb putative export machinery. You will also utilise quantitative PCR and western blotting to analyse gene expression and protein abundance, respectively.

We will use in vitro cell culture to investigate if transfer of vtRNA via uptake of vault particles can endow an apoptosis resistant phenotype in cells that would be found in the TME. Vault particles will be tagged by genetic manipulation to allow tracking of release and uptake.

Novelty and impact

Many research groups’ efforts are focused on the role of EVs in the TME. However, other extracellular particles are largely neglected. To our knowledge, this would be the first study to focus on the vault particle as a novel mediator of intercellular communication.

Supervisor one: Dr Stuart Hunt

https://www.sheffield.ac.uk/dentalschool/our-people/academic-staff/stuart-hunt

https://twitter.com/Dr_Stuart_Hunt

Supervisor two: Prof Dan Lambert

https://www.sheffield.ac.uk/dentalschool/our-people/academic-staff/daniel-lambert

Benefits of being in the DiMeN DTP:

This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle, York and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.

We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.

Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: https://www.dimen.org.uk/blog

Further information on the programme and how to apply can be found on our website:

https://www.dimen.org.uk/how-to-apply