The overall hypothesis guiding our work is that the core defect, impaired anion transport due to missing or defective CFTR, compromises the volume and composition of airway surface liquid (ASL) and impedes mucociliary clearance, leading to infection, cellular stress and inflammation. Studies of channel function, biogenesis and drug-mediated correction, performed in polarized HBE cells, are predictive of clinical outcome in the pipeline of therapeutic strategies, as demonstrated by the development of VX-770 and VX-809.
Goal: Provide translationally relevant physiological assays of CFTR, ENaC and other CF relevant transport functions to Center investigators
Our five objects:
Protein Biochemistry and Structure Assays
The goal is to utilize new CFTR structure and protein purification information to apply our knowledge of CFTR’s molecular organization to the operations of the cell’s ER quality control and protein folding support system to elucidate and remediate the regulatory pathways and post-translational modifications that impact the biogenesis of CFTR and its mutants. It is now possible to produce milligram quantities of full-length CFTR or its domains, to examine their thermodynamic stabilities, folding efficiencies, functions (e.g. ATP binding and hydrolysis), and to use biophysical methods, and protease protection assays as a measure of their folded state.
Electrophysiologic Assays of Channel Function
The Core performs assays such as Ussing chamber electrophysiology (including permeabilized epithelia), whole-cell and single channel patch clamp, live-cell imaging of halide (MQAE/SPQ), calcium, and pH reporters. Specialized assays, such as the generation of Fluorogen Activated Protein (FAP)-tagged transporters and ion channels, together with medium throughput methods to examine protein trafficking to the plasma membrane or protein itinerary and stability following internalization. Tandem fluorogens provide readout of compartment composition, e.g. pH, Ca, etc. at specific protein locations such as the ASL. The Core provides assistance with protein expression or knockdown in airway cell lines, HBE or HNE using viral vectors. We have also extended the patch clamp technique to HBE cells differentiated on filters with retention of their phenotypic properties for up to 24 hrs.
ASL and Mucus Transport Assays
The Assays Core provides multiple measures of the patho-physiologic properties of HBEs and HNEs, including ciliary beat frequency (CBF), mucociliary transport (MCT), ASL height, ASL viscosity (by FRAP), and ASL pH, for example, as a means of examining the impact of physiological regulators, small molecule modulators or inflammatory cytokines on these determinants of mucociliary clearance.
The Core offers a full range of standard light microscopy methods, but the specialty of the Center for Biologic Imaging (Dr. Simon Watkins, Director) is sophisticated fluorescence microscopy and data analysis, with several live-cell, temperature-controlled stations for quantitative functional measurements. The Center also provides super resolution methods, SIM, STORM and STED for approaches that need to overcome the diffraction limit of conventional light microscopy. Electron microscopy services, transmission, scanning and immune-electron microscopy are also available.
Next-generation Sequencing Assays
To provide a more complete view of the consequences of CFTR loss and the mechanisms of small molecule action, the Core provides methods for the comprehensive analysis of gene expression: RNA-seq, ATAC-seq and single cell RNA-seq. Examples are provided for projects currently underway.
With ion transport as the core defect in CF airways, the successful modulation of CFTR and other channels and transporters impacts properties of the ASL and mucociliary clearance that these assays track in vitro, so as to successfully predict the outcomes of mucus clearance and fluid absorption assays performed in vivo (Core D). This complete approach, initiated in HBEs, establishes the translational efficacy dataset needed for advancing a therapeutic strategy to clinical trials.