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

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Fingerprint Dive into the research topics of 'Real-time fluorescence detection of calcium efflux during vacuolar membrane fusion'. Together they form a unique fingerprint.

  • 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