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Development of high-content fluorescence assays to rapidly monitor CFTR-mediated bicarbonate flow - how do CFTR mutations and modulator drugs affect it?


   UCL Division of Biosciences

   Sunday, December 11, 2022  Funded PhD Project (UK Students Only)

About the Project

1) Generation and optimization of a fluorescence probe to monitor CFTR-mediated anion flow.

The aim is to create a tool to rapidly quantify CFTR-dependent Cl- and HCO3- flow, in the simplified HEK293 expression system. The new assay will be based on a recently developed high-content assay [1, 2]. Cells will be transformed with a bicistronic plasmid encoding a soluble red pH-sensitive fluorescent protein [3] and an N-terminal fusion of CFTR to the I--sensitive yellow fluorescent protein, YFP [4]. Cells expressing these proteins will be grown in 96-well plates, and images will be acquired with a screening microscope equipped with automated fluid-addition robotics. CFTR will be pre-activated in HCO3- -free vs. Cl- -free solutions. Once steady-state activation is reached, a second addition will increase extracellular I-. By monitoring quenching of YFP over time we will compare Cl-/I- vs. HCO3-/I- exchange. Fluorescence timelines following I- addition will be fit using a mathematical model describing transmembrane ionic fluxes and intracellular and extracellular pH and buffers. Finally, the average red fluorescence throughout the cell (following cytosolic alkalinisation) will be used as a unit, to quantify how much CFTR is in proximity of the membrane. The assay will allow simultaneous monitoring of multiple readouts (CFTR Cl- and HCO3- conductance, CFTR membrane proximity), at the level of individual cells, while automated image analysis will extract information from thousands of cells.

2) Generation and optimization of a cellular anion flux biosensor system

Results from expression and inhibitor studies (carried out in other SRC labs exploiting ex-vivo material), will help identify components likely to be necessary to recapitulate recent results: while bile ducts expressing WT-CFTR had a high HCO3- selectivity, CFTR modulator treatment of F508del-CFTR-expressing ducts increased Cl- but not HCO3- secretion [5]. What protein(s) do we need to coexpress in order to recover the cholangiocyte effect in HEK293 cells? By incorporating transport and regulatory proteins, we propose to build a relevant “minimal model” biosensor HEK293 cell-line. There is evidence to support the feasibility of this approach: coexpression of CFTR and the protein kinase WNK1, alone, was sufficient to reconstruct a system capable of modulating CFTR anion selectivity [6]. Mechanistic insight will emerge from building the system from its component parts.

3) Characterization of Cl- vs. HCO3- fluxes in mutant CFTR variants, before and after treatment with CFTR modulator drugs

The minimal model biosensor system will be used to profile how a panel of 62 CF-causing CFTR mutations [2] affect anion fluxes. Further, for each mutant, the effect of 20 hours’ treatment with modulators will be quantified. How do modulators affect Cl- and HCO3- transmembrane flow? Is there any mutant/modulator combination that differentially recovers one or the other? The empirical screens will provide material for generating hypotheses, and pinpoint compounds/residues/conditions requiring more detailed investigations in native systems (to be carried out in other SRC labs).

Intense cross-discipline communication and iterative interactions with other members of the SRC team will enable a deeper understanding of how CFTR-mediated anion fluxes are modulated.

The PhD studentship is available to start in September 2023. Please contact Dr Paola Vergani () for informal enquiries.

You can view more PhD studentships here: https://www.findaphd.com/phds/program/4-fully-funded-interdisciplinary-phd-studentships/?p5828


Funding Notes

As well as funding materials, consumables and use of facilities, the award provides a generous tax-free stipend (£24,093, £26,057 and £26,839 per annum during the PhD years) and covers tuition fees (at home student rates). Funds are also available for attending specialised training workshops and relevant international scientific conferences to increase awareness of scientific advances, but also to provide opportunities for networking and team building.

References

1. Prins, S., Corradi, V., Sheppard, D. N., Tieleman, D. P., and Vergani, P. (2022) Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains. J Biol Chem 298, 101615
2. Prins, S., Langron, E., Hastings, C., Hill, E. J., Stefan, A. C., Griffin, L. D., and Vergani, P. (2020) Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity. J Biol Chem 295, 16529-16544
3. Shen, Y., Rosendale, M., Campbell, R. E., and Perrais, D. (2014) pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis. J Cell Biol 207, 419-432
4. Galietta, L., Haggie, P., and Verkman, A. (2001) Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. FEBS Lett. 499, 220-224
5. Bijvelds, M. J. C., Roos, F. J. M., Meijsen, K. F., Roest, H. P., Verstegen, M. M. A., Janssens, H. M.et al., . . . de Jonge, H. R. (2022) Rescue of chloride and bicarbonate transport by elexacaftor-ivacaftor-tezacaftor in organoid-derived CF intestinal and cholangiocyte monolayers. J Cyst Fibros 21, 537-543
6. Kim, Y., Jun, I., Shin, D. H., Yoon, J. G., Piao, H., Jung, J.et al., . . . Lee, M. G. (2020) Regulation of CFTR Bicarbonate Channel Activity by WNK1: Implications for Pancreatitis and CFTR-Related Disorders. Cell Mol Gastroenterol Hepatol. 9, 79-103

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