Monitoring bile acid transport in single living cells using a genetically encoded Förster resonance energy transfer sensor

Abstract

Bile acids are pivotal for the absorption of dietary lipids and vitamins and function as important signaling molecules in metabolism. Here, we describe a genetically encoded fluorescent bile acid sensor (BAS) that allows for spatiotemporal monitoring of bile acid transport in single living cells. Changes in concentration of multiple physiological and pathophysiological bile acid species were detected as robust changes in F€ orster resonance energy transfer (FRET) in a range of cell types. Specific subcellular targeting of the sensor demonstrated rapid influx of bile acids into the cytoplasm and nucleus, but no FRET changes were observed in the peroxisomes. Furthermore, expression of the liver fatty acid binding protein reduced the availability of bile acids in the nucleus. The sensor allows for single cell visualization of uptake and accumulation of conjugated bile acids, mediated by the Na 1 -taurocholate cotransporting protein (NTCP). In addition, cyprinol sulphate uptake, mediated by the putative zebrafish homologue of the apical sodium bile acid transporter, was visualized using a sensor based on the zebrafish farnesoid X receptor. The reversible nature of the sensor also enabled measurements of bile acid efflux in living cells, and expression of the organic solute transporter ab (OSTab) resulted in influx and efflux of conjugated chenodeoxycholic acid. Finally, combined visualization of bile acid uptake and fluorescent labeling of several NTCP variants indicated that the sensor can also be used to study the functional effect of patient mutations in genes affecting bile acid homeostasis. Conclusion: A genetically encoded fluorescent BAS was developed that allows intracellular imaging of bile acid homeostasis in single living cells in real time. (HEPATOLOGY 2013;57:740-752) B ile acids operate as signaling molecules with systemic endocrine functions that are very important for lipid, glucose, and energy homeostasis and are essential for intestinal absorption of dietary fat and fat-soluble vitamins.

Key discoveries in bile acid chemistry and biology and their clinical applications: history of the last eight decades

“…The molecules that constitute the circulating pool a bile acid binding site (based on the binding site of FXR) and two reporter molecules whose fl uorescence is determined by the extent to which bile acids occupy the binding site, the greater the binding, the less the fl uorescence. The technique, as described by van der Velden et al ( 217 ), uses the well-known Forster resonance energy transfer (FRET) principle, and has already been used to characterize bile acid transport into isolated hepatocytes.…”

Ablating L-FABP in SCP-2/SCP-x null mice impairs bile acid metabolism and biliary HDL-cholesterol secretion

“…L-FABP also bound other oxidized lipids to protect hepatic cells from oxidative stress (58,85,86,89,90). L-FABP inhibits microsomal sulfation of select bile acids in vitro (70), is essential for bile acid uptake and intracellular transport as evidenced by photoaffinity-labeled bile acid cross-linking studies (15), and decreases bile acid distribution into some intracellular compartments (82). Thus, by binding bile acids in the cytoplasm, L-FABP may act to retain bile acids within the hepatocytes and decrease their excretion into bile via export pumps that concentrate bile acids Ͼ1,000-fold in bile compared with hepatocytes (43).…”

“…While SCP-2 also binds cholesterol hydroperoxides, it facilitates, rather than prevents, the toxic effects of these hydroperoxides (39 -41, 84). Analogous to the effects of L-FABPbinding bile acids in the cytosol to reduce bile acid distribution to the nucleus (82), by binding oxysterols the SCP-2 may sequester these ligands in the cytosol and peroxisomes to decrease their availability in the nucleus. This may explain in part why loss of SCP-2/SCP-x increased expression of Cyp7A1 and Cyp27A1, despite unaltered levels of LXR␣.…”

Single-molecule FRET for Ultrasensitive Detection of Biomolecules

“…Recently, Itoh et al developed a smFRET method to monitor the local balance between the guanine nucleotide exchange factors and the GTPase-activating proteins for Rac1 and Cdc42 on the membrane of living cells [92]. In addition, van der Velden et al demonstrated a genetically encoded fluorescent bile acid sensor for spatio-temporal monitoring the transportation of bile acid in single living cell [93]. Tanimura …”