Amplification of a FRET Probe by Lipid-Water Partition for the Detection of Acid Sphingomyelinase in Live Cells.
Real-time monitoring of acid sphingomyelinase (ASM) activity is crucial for investigating its role in lipid-mediated signaling processes. In this study, we synthesized fluorescent phosphosphingolipids capable of FRET by phosphorodichloridate chemistry. These sphingomyelin analogues are substrates for recombinant human ASM and can be used to monitor ASM activity by fluorescence spectroscopy. Incubation with cell lysates from wild-type and knock-out mice further confirmed probe cleavage to be exclusive to ASM. We also systematically exploited the environmental sensitivity of the fluorophores to achieve significant increases in responsiveness. This concept may be transferred to other lipid probes in the future. The ASM activity in live cells was imaged by two-photon-excitation microscopy.
Institute of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489, Berlin, Germany.
Thomas
T
Pinkert
Institute of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489, Berlin, Germany.
David
D
Furkert
Institute for Biology and IRI Lifesciences, Humboldt-Universität zu Berlin, Invalidenstrasse 42, 10115, Berlin, Germany.
Thomas
T
Korte
Institute for Biology and IRI Lifesciences, Humboldt-Universität zu Berlin, Invalidenstrasse 42, 10115, Berlin, Germany.
Andreas
A
Herrmann
Institute of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489, Berlin, Germany.
Christoph
C
Arenz
D000818:Animals; D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry*; D051379:Mice; D018345:Mice, Knockout; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis* / Q000378:metabolism; D013997:Time Factors; D014867:Water / Q000737:chemistry*
D005456:Fluorescent Dyes; D008055:Lipids; D011994:Recombinant Proteins; D014867:Water; D013108:Sphingomyelin Phosphodiesterase
FRET probes; enzymes; fluorescence spectroscopy; membranes; sphingolipids