Isolation of yeast mutants defective for localization of vacuolar vital dyes.
Isolation of yeast mutants defective for localization of vacuolar vital dyes.
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DOI：
10.1073/pnas.95.20.11721
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发表时间：
1998-09
影响因子：
11.1
通讯作者：
Bing Zheng;Jennifer N. Wu;Wendy Schober;Dorothy E. Lewis;Thomas A. Vida
中科院分区：
文献类型：
--
作者：
Bing Zheng;Jennifer N. Wu;Wendy Schober;Dorothy E. Lewis;Thomas A. Vida
流式细胞分选的应用是用于分离酿酒酵母突变体错定位液泡重要染料。该筛选基于亲脂性苯基化合物N-（3-三乙基氨丙基）-4-（6-（4-（二乙基氨基）苯基）六己基炔基）二溴吡啶（FM4-64）在15度下标记从质膜到液泡膜的内吞噬中间体的能力。fm3 -64和羧基二氯荧光素（一种液泡腔生命染色）在15度下染色的细胞，与在30度或38度下染色的细胞相比，红/绿荧光有明显的变化。基于这种特征转移的流式细胞术选择允许分离16个突变体。这包括12个互补基团，我们将其命名为SVL，用于苯乙烯基染料液泡定位。这些人被分成三个班。第一类突变体含有非常大的液泡；II类突变体有非常破碎的液泡；III类突变体在FM4-64点状/弥漫性染色中显示最强的svl表型。与先前分离的突变体，即svl2/vps41、svl6/vps16和svl7/fab1，观察到有限的遗传重叠。其余的svl突变体似乎代表了新的基因，其中两个表现出对温度敏感的液泡染色形态。另一个突变体sv18在磷脂酰胆碱和磷脂酰乙醇胺的吸收和分选方面表现出缺陷。我们的流式细胞术策略可能有助于分离其他突变体，其中荧光化合物的错误定位可以检测到。
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.