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. 1998 Sep 29;95(20):11721-6.
doi: 10.1073/pnas.95.20.11721.

Isolation of yeast mutants defective for localization of vacuolar vital dyes

B Zheng  1 , J N Wu, W Schober, D E Lewis, T Vida
Affiliations

Isolation of yeast mutants defective for localization of vacuolar vital dyes

B Zheng et al. Proc Natl Acad Sci U S A. .

Abstract

An application of flow cytometric sorting is used for isolation of Saccharomyces cerevisiae mutants that mislocalize vacuolar vital dyes. This screen is based on the ability of a lipophilic styryl compound, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrie nyl )pyridinium dibromide (FM4-64), to label endocytic intermediates from the plasma membrane to the vacuole membrane at 15 degreesC. Cells stained at 15 degreesC for both FM4-64 and carboxydichlorofluorescein diacetate (a vacuolar luminal vital stain), had a pronounced shift in red/green fluorescence from cells stained at 30 degrees or 38 degreesC. Flow cytometric selection based on this characteristic shift allowed the isolation of 16 mutants. These comprised 12 complementation groups, which we have designated SVL for styryl dye vacuolar localization. These groups were put into three classes. Class I mutants contain very large vacuoles; class II mutants have very fragmented vacuoles; and class III mutants show the strongest svl phenotype with punctate/diffuse FM4-64 staining. Limited genetic overlap was observed with previously isolated mutants, namely svl2/vps41, svl6/vps16, and svl7/fab1. The remaining svl mutants appear to represent novel genes, two of which showed temperature-sensitive vacuole staining morphology. Another mutant, svl8, displayed defects in uptake and sorting of phosphatidylcholine and phosphatidylethanolamine. Our flow cytometric strategy may be useful for isolation of other mutants where mislocalization of fluorescent compounds can be detected.

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Figures

Figure 1
Figure 1
Cellular energy is required for localization of FM4–64 to the vacuolar membrane from endosomal intermediates. Diploid yeast cells were stained with FM4–64 at 15°C for 30 min in rich media containing glucose (2%). The cells were placed on ice, washed twice with rich media (no glucose), and then incubated at 30°C for 30 min with glucose (2%) or NaN3 and NaF (10 mM each), as indicated. The cells were then viewed with epifluorescence microscopy by using a Texas red filter set and DIC optics. Arrows point to punctate intermediates and vacuole membrane staining. (Bar = 5 μm.)
Figure 2
Figure 2
Flow cytometry can distinguish cells with punctate FM4–64 staining morphology from cells with vacuolar membrane staining morphology. Diploid yeast cells were stained with FM4–64 (10 μM) and CDCFDA (10 μM) at the indicated temperatures. At 30°C/38°C (A), the cells were pulsed with FM4–64 for 30 min and chased for 60 min. After 60 min, CDCFDA was added (at pH 4) for 15 min. At 15°C (B), the cells were simultaneously pulsed with both FM4–64 and CDCFDA (50 μM) for 30 min. For flow cytometry, haploid yeast cells (SEY6210) were stained with CDCFDA alone (C), FM4–64 alone at 30°C (D), and 15°C (E), or stained with both CDCFDA and FM4–64 at 38°C (F) or 15°C (G). The fluorescence emission was simultaneously monitored at 525 (green) and 590 (red) nm. After overlaying the patterns from double-staining cells at 38° and 15°C, a sort window was defined for putative mutants. Each flow cytometry sample represents ≈2–5 × 104 cells. The white and light gray areas represent the most and least events, respectively.
Figure 3
Figure 3
Morphology of the svl mutants. (A) Yeast cells from wild-type, SVL, or from svl2, svl7, and svl8 cultures were double-stained with CDCFDA and FM4–64 at 25°C, as indicated. Images of the cells were captured with epifluorescence microscopy separately by using a fluorescein isothiocyanate (FITC) or Texas red filter set, and DIC optics, as indicated. The overlay represents a digital blending of the two separate fluorescence captures. The inset in the svl2 panel represents typical vacuole morphology in vps17Δ cells. [Bar = 5 μm (Inset = 2.5 μm).] (B) SVL and svl 8 strains were grown to early-log phase. Cells were stained with BODIPY-PC and BODIPY-PE, and examined by epifluorescence microscopy, as indicated. (Bar = 5 μm.)
Figure 4
Figure 4
ts staining with vacuolar vital dyes in two svl mutants. (A) Yeast cells from wild-type (SVL) and the svl3–1 mutant were stained at 25°C or after a 60-min preincubation at 38°C with CDCFDA and quinacrine for 15 min, as indicated. The cells were then viewed with epifluorescence microscopy using a fluorescein isothiocyanate filter set and DIC optics. (B) Cells from the svl12–1 mutant were double-stained with CDCFDA and FM4–64 at 25°C or after a 60 min preincubation at 38°C. Images of the cells were captured with epifluorescence microscopy separately using a fluorescein isothiocyanate, or Texas red filter set, and DIC optics, as indicated. (Bar = 5 μm.)
Figure 5
Figure 5
Flow cytometry of three representative svl mutants. Yeast cells from wild-type, SVL, or the svl12, svl2, and svl3 mutants were double-stained with CDCFDA and FM4–64 after a 60-min preincubation at 38°C, as indicated. The cells were then subjected to flow cytometry using the same conditions as Fig. 2. The white and dark gray areas represent the most and least events, respectively. Representative cells from each sample are shown with digital blending of the two separate fluorescein isothiocyanate and Texas Red filter sets plus DIC optics. The bottom panel is an overlay representing >75% of the population from each strain. (Bar = 5 μm.)
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