Assessment of FUN-1 vital dye staining: Yeast with a block in the vacuolar sorting pathway have impaired ability to form CIVS when stained with FUN-1 fluorescent dye

Brandin D. Essary, Pamela Marshall

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide] is a fluorescent dye used in studies of yeast and other fungi to monitor cell viability in the research lab and to assay for active fungal infection in the clinical setting. When the plasma membrane is intact, fungal cells internalize FUN-1 and the dye is seen as diffuse green cytosolic fluorescence. FUN-1 is then transported to the vacuole in metabolically active wild type cells and subsequently is compacted into fluorescent red cylindrical intravacuolar structures (CIVS) by an unknown transport pathway. This dye is used to determine yeast viability, as only live cells form CIVS. However, in live Saccharomyces cerevisiae with impaired protein sorting to the yeast vacuole, we report decreased to no CIVS formation, depending on the cellular location of the block in the sorting pathway. Cells with a block in vesicle-mediated transport from the Golgi to prevacuolar compartment (PVC) or with a block in recycling from the PVC to the Golgi demonstrate a substantial impairment in CIVS formation. Instead, the FUN-1 dye is seen either in small punctate structures under fluorescence or as diffuse red cytosol under white light. Thus, researchers using FUN-1 should be cognizant of the limitations of this procedure in determining cell viability as there are viable yeast mutants with impaired CIVS formation.

Original languageEnglish (US)
Pages (from-to)208-212
Number of pages5
JournalJournal of Microbiological Methods
Volume78
Issue number2
DOIs
StatePublished - Aug 1 2009

Keywords

  • FUN-1 dye
  • False negative
  • Saccharomyces cerevisiae
  • Vacuolar protein sorting
  • Vps mutant

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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