John W. Hanrahan

Hypoxia is associated with a range of maladies, inflammation, and impaired immunity. The airway epithelial barrier contends with constant exposure to microbes but can be weakened with hypoxia and diseases, such as cystic fibrosis (CF). People with CF (pwCF) have defective cystic fibrosis transmembrane conductance regulator (CFTR) function leading to reduced immune function, excess mucous accumulation, and chronic infection. CFTR is a cAMP‐dependent anion channel that is regulated in part by adenylyl cyclase 6 (AC6). G protein‐coupled receptors (GPCRs) such as the chemosensory bitter taste receptors (T2Rs) have been shown to alter inhibitory G proteins and cAMP levels. T2Rs also mediate innate immunity responses and detect quorum sensing molecules (QSMs) through T2R14. The impact of hypoxia on these processes, in human airways, has not yet been characterized. We analyzed protein expression and functional endpoints at normal (21%), mild (10%), and severe (1%) oxygen levels to establish the effects of hypoxia on these processes in human bronchial epithelial cells. Our results show that severe hypoxia leads to decreased AC6 expression without altering Gαi/Gαs/T2R14 compared to wild‐type controls. Hypoxia induced ligand and oxygen dependent effects on T2R14 functional responses to fungal QSMs, farnesol, and tyrosol. IL‐5 release was increased in QSM treated CF cells at 1% oxygen. Severe hypoxia inhibited forskolin‐induced currents due to CFTR and reduced cAMP. These results demonstrate expression level and functional alterations due to hypoxia in airway epithelia, including evidence that reduced AC6 expression and function in severe hypoxia is associated with CFTR dysfunction, establishing a potential link between these proteins and the functional outcome of airway epithelial response to hypoxia.

Current models of the cell membrane assume a heterogenous environment such as sphingomyelin and cholesterol-enriched nano- and microdomains, which are thought to functionally sequester proteins. Besides lipid-ordered domains, membrane proteins can interact with protein complexes by transient binding—necessary for their functional role. Here, we show that an extension of k-space Image Correlation Spectroscopy applied to standard fluorescence microscopy image time series can be used to characterize the protein confinement in heterogeneous membranes. To validate this method, we simulated confined diffusion of tracer particles in a system of static microdomains where we varied the domain size, domain density, confinement probability and diffusion coefficients of tracer particles. We show how the kICS correlation function changes with these parameters and gives rise to emergent properties of the system such as apparent domain sizes and characteristic diffusion coefficients. As a validity check, we apply this analysis to study the dynamics of lipid domain-associated glycosylphosphatidyl inositol (GPI)-anchored proteins labeled by green fluorescent proteins (GPI-GFP) in intact COS-7 cell membranes, and upon domain-disrupting enzyme treatments.

Single-stranded messenger ribonucleic acid (mRNA) plays a pivotal role in transferring genetic information, and tremendous effort has been devoted over the years to utilize its transcription efficacy in therapeutic interventions for a variety of diseases with high morbidity and mortality. Lipid nanocarriers have been extensively investigated for mRNA delivery and enabled the rapid and successful development of mRNA vaccines against SARS-CoV-2. Some constraints of lipid nanocarriers have encouraged the development of alternative delivery systems, such as polymer-based soft nanoparticles, which offer a modular gene delivery platform. Such macromolecule-based nanocarriers can be synthetically articulated for tailored parameters including mRNA protection, loading efficacy, and targeted release. In this review, we highlight recent advances in the development of polymeric architectures for mRNA delivery, their limitations, and the challenges that still exist, with the aim of expediting further research and the clinical translation of such formulations.

CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.

CFTR (cystic fibrosis transmembrane conductance regulator) is a tightly regulated anion channel that mediates chloride and bicarbonate conductance in many epithelia and in other tissues, but whether its regulation varies depending on the cell type has not been investigated. Epithelial CFTR expression is highest in rare cells called ionocytes. We studied CFTR regulation in control and ionocyte-enriched cultures by transducing bronchial basal cells with adenoviruses that encode only eGFP or FOXI1 (forkhead box I1) + eGFP as separate polypeptides. FOXI1 dramatically increased the number of transcripts for ionocyte markers ASCL3 (Achaete-Scute Family BHLH Transcription Factor 3), BSND, ATP6V1G3, ATP6V0D2, KCNMA1, and CFTR without altering those for secretory (SCGB1A1), basal (KRT5, KRT6, TP63), goblet (MUC5AC), or ciliated (FOXJ1) cells. The number of cells displaying strong FOXI1 expression was increased 7-fold, and there was no evidence for a broad increase in background immunofluorescence. Total CFTR mRNA and protein levels increased 10-fold and 2.5-fold, respectively. Ionocyte-enriched cultures displayed elevated basal current, increased adenylyl cyclase 5 expression, and tonic suppression of CFTR activity by the phosphodiesterase PDE1C, which has not been shown previously to regulate CFTR activity. The results indicate that CFTR regulation depends on cell type and identifies PDE1C as a potential target for therapeutics that aim to increase CFTR function specifically in ionocytes.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death and cigarette smoke is the main risk factor. Detecting its earliest stages and preventing a decline in lung function are key goals. The pathogenesis of COPD is complex but has some similarities to cystic fibrosis (CF), a disease caused by mutations in the cftr gene. CF leads to chronic inflammation, abnormal mucus, and cycles of infection. Cigarette smoke exposure also causes CFTR dysfunction, and it is probably not a coincidence that inflammation, mucus obstruction, and infections are also characteristics of COPD, although the exacerbations can be quite different. We review here the acute effects of cigarette smoke on CFTR function and its potential role in COPD. Understanding CFTR regulation by cigarette smoke may identify novel drug targets and facilitate the development of therapeutics that reduce the progression and severity of COPD.

Membrane proteins often cluster in nanoscale membrane domains (lipid rafts) that coalesce into ceramide-rich platforms during cell stress, however the clustering mechanisms remain uncertain. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis (CF), forms clusters that are cholesterol dependent and become incorporated into long-lived platforms during hormonal stimulation. We report here that clustering does not involve known tethering interactions of CFTR with PDZ domain proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are abolished by the disease mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate CF therapeutics that correct mutant protein folding restore both trafficking and normal lipid interactions in the plasma membrane. This article has an associated First Person interview with the first author of the paper.

Although some therapeutic progress has been achieved in developing small molecules that correct F508del-CFTR defects, the mechanism of action (MoA) of these compounds remain poorly elucidated. Here, we investigated the effects and MoA of MCG1516A, a newly developed F508del-CFTR corrector. MCG1516A effects on wild-type (WT) and F508del-CFTR were assessed by immunofluorescence microscopy, and biochemical and functional assays both in cell lines and in intestinal organoids. To shed light on the MoA of MCG1516A, we evaluated its additivity to the FDA-approved corrector VX-661, low temperature, genetic revertants of F508del-CFTR (G550E, R1070W, and 4RK), and the traffic-null variant DD/AA. Finally, we explored the ability of MCG1516A to rescue trafficking and function of other CF-causing mutations. We found that MCG1516A rescues F508del-CFTR with additive effects to VX-661. A similar behavior was observed for WT-CFTR. Under low temperature incubation, F508del-CFTR demonstrated an additivity in processing and function with VX-661, but not with MCG1516A. In contrast, both compounds promoted additional effects to low temperature to WT-CFTR. MCG1516A demonstrated additivity to genetic revertant R1070W, while VX-661 was additive to G550E and 4RK. Nevertheless, none of these compounds rescued DD/AA trafficking. Both MCG1516A and VX-661 rescued CFTR processing of L206W- and R334W-CFTR with greater effects when these compounds were combined. In summary, the absence of additivity of MCG1516A to genetic revertant G550E suggests a putative binding site for this compound on NBD1:NBD2 interface. Therefore, a combination of MCG1516A with compounds able to rescue DD/AA traffic, or mimicking the actions of revertant R1070W (e.g., VX-661), could enhance correction of F508del-CFTR defects.

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.