The cystic fibrosis transmembrane conductance regulator, CFTR, is a plasma membrane anion channel which plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrhoeas, while CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR – one boosting ion-channel function (potentiators), the other increasing plasma membrane density (correctors) – have provided significant health benefits to CF patients. Here we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein’s proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease.
In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name “neighbourhood watch” model) illuminates the classic “French Flag Problem” and how positional information is interpreted by a sheet of cells in a large developing system.
We have isolated and sequenced two full-length cDNA clones encoding actin from carrot. The two carrot clones are almost identical at the nucleotide level, and are quite homologous to each other and to other plant actins at the amino acid level. In those regions where amino acid variation exists between the two genes from carrot, the differences have arisen from very simple changes at the nucleotide level. The most common changes are nucleotide insertion(s) coupled to the deletion of a different nucleotide(s) nearby in the DNA sequence, resulting in the restoration of the proper reading frame for the protein; thus, these changes can be viewed as multiple or coupled frameshift mutations. There are almost no base substitutions between the two carrot genes. In contrast to this, when the carrot actin nucleotide sequences are compared to those of a soybean actin gene or a maize actin gene, many base substitutions are observed (ca, 21.8% and 23.5%), more than half of which are third base changes which do not alter the protein sequence. At the amino acid level, both carrot genes show greater similarity to maize actin than they do to soybean actin, thus reinforcing the idea that plant actin genes diverged from a single common ancestral actin gene prior to the divergence of monocots and dicots.
18CFTR mutations cause cystic fibrosis by affecting how many channels reach the membrane, 19 and/or how well they work. This study presents an assay that simultaneously measures CFTR 20 biogenesis and function. A screen of 62 disease-causing mutations provides clues on how 21 approved drug VX-770 works.Abstract 24 Cystic fibrosis (CF) is a life-limiting disease caused by mutations in the CFTR gene, 25 encoding a plasma membrane anion-selective channel. Because CF-causing mutations affect 26 both CFTR permeation/gating and biogenesis, multi-assay approaches have been implemented 27 in drug development, sequentially screening for channel function and membrane density. Here 28 we present the first assay capable of simultaneous assessment of both CFTR characteristics. 29To validate our assay, we investigate F508del-CFTR, the most common disease-30 causing mutant, confirming rescue by incubation at low temperature, treatment with CFTR-31 targeting drugs and introduction of second-site revertant mutation R1070W. In addition, we 32 characterize a panel of 62 CF-causing mutations and profile effects of acute treatment with 33 approved drug VX-770 (ivacaftor). Measurements using the rare mutation panel correlate well 34 with published results, further validating the assay. Furthermore, mapping the potentiation 35 profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-36 770 might allow an open-channel conformation with an alternative arrangement of domain 37 interfaces around site 1. 38The assay is a powerful tool for investigation of CFTR ion channel biophysics, allowing 39 rapid and accurate inferences on gating/permeation properties and on how these are affected 40 by mutations and compounds. By providing a two-dimensional molecular characterization of 41 mutant CFTR proteins, our assay can better inform development of therapies tailored for 42 individual CFTR variants. Finally, the integrated assay boosts the potential for discovery of 43 dual-acting compounds, simultaneously repairing both biogenesis and function. 44 45 Keywords: protein transport, fluorescence imaging, precision medicine, VX-770.46 3 Non-standard Abbreviations 47 ABC ATP-binding cassette 48 CF Cystic Fibrosis 49 CFTR Cystic Fibrosis Transmembrane Conductance Regulator 50 FmCherry cell average normalized mCherry fluorescence intensity over the entire cell 51 FYFP cell average normalized YFP fluorescence intensity over the entire cell 52 FYFP membrane average normalized YFP fluorescence intensity within the membrane zone 53 GCFTR CFTR conductance 54 Gtrans transient anion conductance 55 IRES internal ribosome entry site 56 NBD nucleotide binding domain 57 PDL poly-D-lysine 58 PO open probability 59 CFTR membrane density, as defined in this paper 60 SSR sum of squared residuals 61 trans time constant of the transient anion conductance 62 TM transmembrane helix 63 VM membrane potential 64 WT wild type 65 YFP yellow fluorescent protein 66 67 Cystic fibrosis (CF) is a common life-limiting genetic disease. Alth...
Classical studies have established that the marginal zone, a ring of extraembryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extraembryonic region (area opaca) was only thought to have nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region: first, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extraembryonic tissues in early development.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.