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  and an N-terminal fusion of CFTR to the I--sensitive yellow fluorescent protein, YFP . 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 . 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 . 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  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 (firstname.lastname@example.org) for informal enquiries.
You can view more PhD studentships here: https://www.findaphd.com/phds/program/4-fully-funded-interdisciplinary-phd-studentships/?p5828