Real time monitoring of intracellular bile acid dynamics using a genetically encoded FRET-based Bile Acid Sensor

Förster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell

IgG4+ B-Cell receptor clones distinguish IgG4-related disease from primary sclerosing cholangitis and biliary/pancreatic malignancies.

IgG4-related disease of the biliary tree and pancreas is difficult to distinguish from sclerosing cholangitis and biliary/pancreatic malignancies. An accurate noninvasive test for diagnosis and monitoring of disease activity is lacking. We demonstrate that dominant IgG4+ B-cell receptor (BCR) clones determined by nextgeneration sequencing (NGS) accurately distinguish patients with IgG4-associated cholangitis/autoimmune pancreatitis (n=34) from those with primary sclerosing cholangitis (n=17) and biliary/pancreatic malignancies (n=17). A novel, more affordable, widely applicable quantitative polymerase chain reaction (qPCR) protocol analyzing the IgG4/IgG RNA-ratio in blood, also achieves excellent diagnostic accuracy (n=125). Moreover, this qPCR-test performed better than serum IgG4 levels in sensitivity (94% versus 86%) and specificity (99% versus 73%), and correlates with treatment response (n=20). Conclusion: IgG4+ BCR clones and IgG4/IgG RNA-ratiomarkedly improve delineation, early diagnosis and monitoring of IgG4-related disease of the biliary tree and pancreas.</p

A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel–Palade body exocytosis in endothelial cells

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated WPB exocytosis critically depends on their proper recruitment to the plasma membrane, but factors involved in WPB-plasma membrane tethering are not known. Here we identify Munc13-4, a protein mutated in familial hemophagocytic lymphohistiocytosis 3, as a WPB-tethering factor. Munc13-4 promotes histamine-evoked WPB exocytosis and is present on WPBs, and secretagogue stimulation triggers an increased recruitment of Munc13-4 to WPBs and a clustering of Munc13-4 at sites of WPB-plasma membrane contact. We also identify the S100A10 subunit of the annexin A2 (AnxA2)-S100A10 protein complex as a novel Munc13-4 interactor and show that AnxA2-S100A10 participates in recruiting Munc13-4 to WPB fusion sites. These findings indicate that Munc13-4 supports acute WPB exocytosis by tethering WPBs to the plasma membrane via AnxA2-S100A10