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(BBSRC DTP CASE) Analysing the efficiency of protein secretion using Lattice Lightsheet Microscopy


Project Description

Protein-based biopharmaceuticals are of tremendous importance both for providing disease treatments, and for the UK economy. They are typically manufactured using mammalian host cells, but some are much harder to produce than others. Such proteins may be delayed in the secretory pathway due to mis-folding and aggregation in the endoplasmic reticulum (ER), slow ER exit, or may be held up in the Golgi apparatus. Each protein may have different problems, and therefore may require distinct solutions to enhance production.
Live cell imaging offers an ideal way to identify where such problems occur, as a wave of traffic through the secretory pathway can be visualised. Importantly, such single cell analysis would reveal if individual cells within the population behave consistently or heterogeneously, and would provide rich data that define the spatial and temporal dynamics of secretion.
This project will apply advanced live cell imaging using the 3i Lattice Lightsheet Microscope (LLSM) to reveal how ‘easy’ and ‘difficult’ cargo proteins are trafficked through the secretory pathway in different cell types. The LLSM, designed by Nobel Prize winner Eric Betzig, is revolutionising live cell imaging in 4D by virtue of its speed, resolution and the minimal photodamage it causes. As part of the Ph.D., a placement with 3i in Denver, Colorado, will involve building, aligning, testing and running an LLSM.
To assess trafficking, the secretory proteins of interest will be tagged with a photo-switchable probe, which will allow a “pulse” of labelled protein to be visualised as it transits from the ER, through the Golgi apparatus, and on to the plasma membrane. Machine learning approaches will be developed to quantitate the rate of transport through the secretory pathway. We will establish new methods for mounting a range of live cells on the LLSM in orientations which optimise secretory pathway visualisation in a range of cell samples. This will involve nanofabrication and development of different chamber designs.
This is a truly inter-disciplinary project led by a team comprising two cell biologists, a materials scientist and 3i microscopy applications specialists. This breadth and depth of interdisciplinary research would be hard to find in a project set in a single laboratory. The project will equip the student with a range of versatile skills including cell culture, advanced light microscopy and nanofabrication. The skills and knowledge provided by project will provide an excellent foundation for a future career in the biosciences, imaging and analysis, or materials science.

https://www.research.manchester.ac.uk/portal/viki.allan.html
https://www.research.manchester.ac.uk/portal/martin.p.lowe.html
https://www.research.manchester.ac.uk/portal/sarah.cartmell.html

Entry Requirements:
Applications are invited from UK/EU nationals only. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

Funding Notes

This project is to be funded under the BBSRC Doctoral Training Partnership. If you are interested in this project, please make direct contact with the Principal Supervisor to arrange to discuss the project further as soon as possible. You MUST also submit an online application form - full details on how to apply can be found on the BBSRC DTP website View Website

As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.

References

1. Chen et al., 2014. Lattice light-sheet microscopy: imaging molecules to embryos at high temporal and spatial resolution. Science, 346, 1257998.
2. Georgiades et al., 2017. The flexibility and dynamics of the tubules in the endoplasmic reticulum. Scientific Reports, 7, 16474.
3. Witkos et al., 2019. GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation. Nature Commun. 10, 127.
4. Shelley D Rawson, Jekaterina Maksimcuka, Philip J Withers, Sarah H Cartmell X-ray Microscopy; Bringing New Insights to the Life Sciences. accepted by BMC Biology (in press)
5. Madi K, Staines KA, Bay BK, Javaheri B, Geng, H, Bodey AJ, Cartmell S, Pitsillides AA, Lee PD “In situ characterisation of nano-scale strains in a physiologically-representative whole joint loading model” Nature Biomedical Engineering 2019 in press

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