A multimodal iPSC platform for cystic fibrosis drug testing

Cystic fibrosis is a monogenic lung disease caused by dysfunction of the cystic fibrosis transmembrane conductance regulator anion channel, resulting in significant morbidity and mortality. The progress in elucidating the role of CFTR using established animal and cell-based models led to the recent discovery of effective modulators for most individuals with CF. However, a subset of individuals with CF do not respond to these modulators and there is an urgent need to develop novel therapeutic strategies. In this study, we generate a panel of airway epithelial cells using induced pluripotent stem cells from individuals with common or rare CFTR variants representative of three distinct classes of CFTR dysfunction. To measure CFTR function we adapt two established in vitro assays for use in induced pluripotent stem cell-derived airway cells. In both a 3-D spheroid assay using forskolin-induced swelling as well as planar cultures composed of polarized mucociliary airway epithelial cells, we detect genotype-specific differences in CFTR baseline function and response to CFTR modulators. These results demonstrate the potential of the human induced pluripotent stem cell platform as a research tool to study CF and in particular accelerate therapeutic development for CF caused by rare variants

RILP is required for proper morphology and function of late endosomes

Lysosomal degradation of signalling receptors such as the epidermal growth factor (EGF) receptor (EGFR) is an important mechanism for termination of cell signalling. Such degradation involves the endosomal sorting of ubiquitylated receptors into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs) that move along microtubules to fuse with perinuclear lysosomes. The Rab7-interacting lysosomal protein RILP is interesting in this context as it interacts with Vps22 (also known as EAP30) and Vps36 (also known as EAP45), subunits of the endosomal sorting complex required for transport II (ESCRT-II), as well as with the dynein-dynactin motor complex. Because previous functional studies of RILP have been based on its overexpression, we have asked here whether RILP is required for endocytic trafficking of receptors. Depletion of RILP caused elevated levels of four late-endosomal molecules, lyso-bisphosphatidic acid, Lamp1, CD63 and cation-independent mannose-6-phosphate receptors. Electron microscopy showed that endosomes of RILP-depleted cells were morphologically distinct from normal late endosomes and had a strongly reduced content of ILVs. As in Vps22-depleted cells, ligand-mediated degradation of EGFRs was strongly inhibited in RILP-depleted cells, in which endocytosed EGFRs were found to accumulate in early endosomes. By contrast, endocytosis and recycling of transferrin receptors occurred normally in RILP-depleted cells. These results establish that RILP, like the ESCRT proteins, is required for biogenesis of MVEs and degradative trafficking of EGFRs but not for trafficking of transferrin receptors through early endosomes. We propose that RILP might coordinate the biogenesis of MVEs with dynein-mediated motility

IPPI - Integrierte Produkt- und Prozessinnovation - aktuell 1: Staerkung der Innovationsfaehigkeit von Klein- und Mittelbetrieben

SIGLEAvailable from TIB Hannover: QN 290(1)+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman

IPPI - Integrierte Produkt- und Prozessinnovation - aktuell 2: Analysen, Instrumente, Methoden

SIGLEAvailable from TIB Hannover: QN 290(2)+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman

Phage φ29 DNA Replication Organizer Membrane Protein p16.7 Contains a Coiled Coil and a Dimeric, Homeodomain-related, Functional Domain

The Bacillus subtilis phage φ29-encoded membrane protein p16.7 is one of the few proteins known to be involved in prokaryotic membrane-associated DNA replication. Protein p16.7 contains an N-terminal transmembrane domain responsible for membrane localization. A soluble variant lacking the N-terminal membrane anchor, p16.7A, forms dimers in solution, binds to DNA, and has affinity for the φ29 terminal protein. Here we show that the soluble N-terminal half of p16.7A can form a dimeric coiled coil. However, a second domain, located in the C-terminal half of the protein, has been characterized as being the main domain responsible for p16.7 dimerization. This 70-residue C-terminal domain, named p16.7C, also constitutes the functional part of the protein as it binds to DNA and terminal protein. Sequence alignments, secondary structure predictions, and spectroscopic analyses suggest that p16.7C is evolutionarily related to DNA binding homeodomains, present in many eukaryotic transcriptional regulator proteins. Based on the results, a structural model of p16.7 is presented.This work was supported in part by Research Grant 2RO1 GM27242-24 from the National Institutes of Health (to M. S.) and by Grants BIO2003-04445 (to M. G. M.) and BMC2002-03818 (to M. S.) from the Spanish Ministry of Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Peer reviewe