Monitoring bile acid transport in single living cells using a genetically encoded Förster resonance energy transfer sensor.
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 Förster 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(+)-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 αβ (OSTαβ) 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.


CONCLUSIONS
A genetically encoded fluorescent BAS was developed that allows intracellular imaging of bile acid homeostasis in single living cells in real time.
Department of Metabolic Diseases, University Medical Center Utrecht, Utrecht, The Netherlands.
Lieke M
LM
van der Velden
Misha V
MV
Golynskiy
Ingrid T G W
IT
Bijsmans
Saskia W C
SW
van Mil
Leo W J
LW
Klomp
Maarten
M
Merkx
Stan F J
SF
van de Graaf
D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods; D002352:Carrier Proteins; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000096:biosynthesis; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish; D000099150:Receptor, Farnesoid X-Activated
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D018160:Receptors, Cytoplasmic and Nuclear; D027981:Symporters; D000099150:Receptor, Farnesoid X-Activated; C037643:bile acid binding proteins; C476575:organic solute transporter alpha, human; C071908:sodium-bile acid cotransporter