Real-time fluorescence measurement of cell-free endosome fusion: regulation by second messengers.
A quantitative real-time assay of cell-free endosomal vesicle fusion was developed and applied to study fusion mechanisms in endosomes from baby hamster kidney (BHK-21) cells. The assay is based on an irreversible approximately 10-fold increase in BODIPY-avidin fluorescence on binding of biotinylated conjugates. BODIPY-avidin and biotin-dextran were internalized for 10 min at 37 degrees C into separate populations of BHK-21 cells, and endosome fractions were prepared. Postnuclear supernatant fractions underwent ATP- and temperature-dependent fusion, as measured in a sensitive custom-built microfluorimeter by the continuous increase in BODIPY-avidin fluorescence. Fusion processes of efficiency > 2.5% could be detected with 200-ms time resolution in sample volumes of 50 microL containing endosomes derived from approximately 4 x 10(4) cells. The fusion time course consisted of a distinct lag phase (up to 10 min) in which little fusion occurred, followed by an approximately exponential rise (t 1/2 10-30 min; fusion efficiency approximately 15%). The lag phase was reduced by preincubation of separate endosome fractions with ATP at 37 degrees C and by coincubation of endosomes at 22 degrees C before the assay, suggesting a rate-limiting step involving binding of a soluble protein to the endosome membrane. Endosome fusion was strongly inhibited by GTP gamma S, N-ethylmaleimide, and AIF4-. Endosome fusion was not affected by phorbol myristate acetate but was significantly inhibited by cAMP and bovine brain calmodulin. The results establish a sensitive real-time fluorescence assay to quantify the kinetics and extent of endosome fusion in a cell-free system and demonstrate regulation of early endosome fusion by cytosolic second messengers.
Emans
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Department of Medicine, University of California, San Francisco 94143-0521 USA. emans@itsa.ucsf.edu
Verkman
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AS
D000255:Adenosine Triphosphate / Q000378:metabolism; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D001896:Boron Compounds; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D002474:Cell-Free System; D006224:Cricetinae; D000242:Cyclic AMP / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects / Q000502:physiology*; D005033:Ethylmaleimide / Q000494:pharmacology; D005456:Fluorescent Dyes; D005459:Fluorides / Q000494:pharmacology; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D011494:Protein Kinases / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D017607:Aluminum Compounds; D001896:Boron Compounds; D002147:Calmodulin; D005456:Fluorescent Dyes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D005033:Ethylmaleimide; D005459:Fluorides; C032311:aluminum fluoride