Real-time fluorescence detection of calcium efflux during vacuolar membrane fusion

Gregory E. Miner, Rutilio Fratti

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

During in vitro homotypic yeast vacuole fusion Ca2+ is transported into and out of the organelle lumen. In vitro, Ca2+ is taken up from the medium by vacuoles upon the addition of ATP. During the docking stage of vacuole fusion Ca2+ is effluxed from the lumen upon the formation of trans-SNARE complexes between vesicles. Here we describe a real-time fluorescence-based assay to monitor the transport of this cation using purified organelles. Extraluminal Ca2+ is detected when the cation binds the low-affinity fluorescent dye Fluo-4 dextran. This allows for the use of a 96-well microtiter plate to be read in a fluorescence plate reader. Thus, in addition to a curve of calibrated Ca2+ standards, up to 91 experimental conditions can be monitored in a single microplate using this method.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages323-331
Number of pages9
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1860
ISSN (Print)1064-3745

Fingerprint

Membrane Fusion
Vacuoles
Fluorescence
Calcium
Organelles
Cations
SNARE Proteins
Dextrans
Fluorescent Dyes
Adenosine Triphosphate
Yeasts
In Vitro Techniques

Keywords

  • Ca efflux
  • Fluorescence
  • Membrane fusion
  • Membrane trafficking
  • SNARE
  • Yeast vacuole

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Miner, G. E., & Fratti, R. (2019). Real-time fluorescence detection of calcium efflux during vacuolar membrane fusion. In Methods in Molecular Biology (pp. 323-331). (Methods in Molecular Biology; Vol. 1860). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8760-3_21

Real-time fluorescence detection of calcium efflux during vacuolar membrane fusion. / Miner, Gregory E.; Fratti, Rutilio.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 323-331 (Methods in Molecular Biology; Vol. 1860).

Research output: Chapter in Book/Report/Conference proceedingChapter

Miner, GE & Fratti, R 2019, Real-time fluorescence detection of calcium efflux during vacuolar membrane fusion. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1860, Humana Press Inc., pp. 323-331. https://doi.org/10.1007/978-1-4939-8760-3_21
Miner GE, Fratti R. Real-time fluorescence detection of calcium efflux during vacuolar membrane fusion. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 323-331. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8760-3_21
Miner, Gregory E. ; Fratti, Rutilio. / Real-time fluorescence detection of calcium efflux during vacuolar membrane fusion. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 323-331 (Methods in Molecular Biology).
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