TY - JOUR
T1 - Assessment of FUN-1 vital dye staining
T2 - Yeast with a block in the vacuolar sorting pathway have impaired ability to form CIVS when stained with FUN-1 fluorescent dye
AU - Essary, Brandin D.
AU - Marshall, Pamela
N1 - Funding Information:
We thank Joanna M. Tober, Matthew D. Eggleston, and Jayme Gillen for their contributions to the initial stages of this project. BDE was supported in part by Barrett, the Honors College, at Arizona State University. This project was supported in part by a grant from the American Heart Association #0130472T and by a SRCA grant from the New College of Interdisciplinary Arts and Sciences, both to PAM.
Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2009/8
Y1 - 2009/8
N2 - 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.
AB - 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.
KW - FUN-1 dye
KW - False negative
KW - Saccharomyces cerevisiae
KW - Vacuolar protein sorting
KW - Vps mutant
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U2 - 10.1016/j.mimet.2009.05.018
DO - 10.1016/j.mimet.2009.05.018
M3 - Article
C2 - 19501122
AN - SCOPUS:67650127112
SN - 0167-7012
VL - 78
SP - 208
EP - 212
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 2
ER -