A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

TGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways

Author Correction: Organoid culture media formulated with growth factors of defined cellular activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Organoid culture media formulated with growth factors of defined cellular activity

Abstract: The media formulations necessary for deriving and sustaining organoids from epithelial tissues such as prostate, colon, gastric, liver, pancreas, and others have been established. Critical components of organoid media are a set of growth factors that include R-spondins and BMP signalling antagonists such as Noggin or Gremlin 1. Currently, the practical limitations for formulating organoid media of reproducible potency and larger-scale media production that have hampered further technological applications of organoid technology include: the cost of growth factors such as R-spondins and Gremlin 1/Noggin and their production as defined specific activities free of contaminants that may affect organoid growth. Here we report the production of highly pure recombinant Gremlin 1 and R-spondin 1 from bacterial expression for use in organoid media. We detail the workflow for Gremlin 1 and R-spondin 1 expression, purification, quantification of cellular activity, quality control and use in media formulated for culturing organoids derived from a number of tissues. The development of precisely formulated, cost-effective media of defined specific activity will engender the development of novel applications for organoid technology

Author Correction: Organoid culture media formulated with growth factors of defined cellular activity

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Author Correction: Organoid culture media formulated with growth factors of defined cellular activity

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Do silver fir woolly adelgids ( Dreyfusia nordmannianae) facilitate pathogen infestation with Neonectria neomacrospora on Christmas trees ( Abies nordmanniana )?

In Europe, the most widely used Christmas tree species, Abies nordmanniana, has a long history of infestation with the adelgid Dreyfusia nordmannianae. Since 2011, the species has furthermore been increasingly damaged by the fungus Neonectria neomacrospora. The objective was to study whether infestations by N. neomacrospora were facilitated by the presence of adelgids in Nordmann fir and to explore the genetic components of this potential interplay. The damage caused by D. nordmannianae and N. neomacrospora in two Nordmann fir clonal seed orchards in Denmark was evaluated in 2014, and an experimental infestation was conducted to test the performance of Nordmann fir somatic embryogenesis-derived trees exposed to N. neomacrospora and D. nordmannianae individually or in combination by setting up an artificial infestation chamber. The genetic correlation between the two diseases was 0.54 at two clonal seed orchards, indicating that the trees susceptible to N. neomacrospora were also susceptible to D. nordmannianae or vice versa. In the experimental test, only trees in the treatment with both D. nordmannianae and N. neomacrospora developed needle loss and dieback symptoms. Thus, both the field evaluations and the experimental test revealed an interplay between D. nordmannianae and N. neomacrospora damage on Nordmann fir trees. Future studies should explore in more detail how D. nordmannianae interacts with N. neomacrospora. Our findings and future studies on this topic could have important implications for suitable breeding programs for Nordmann fir Christmas trees and forest pest management

A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding.

Funder: Morbus Osler Society Germany Einstein Center for Regenerative Therapies, BerlinTGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways