A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.
A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.
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DOI：
10.1083/jcb.128.5.779
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发表时间：
1995-03
期刊：
影响因子：
--
通讯作者：
Emr SD
中科院分区：
文献类型：
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作者：
Vida TA;Emr SD
我们使用亲脂性苯乙烯基染料 N-(3-三乙基铵丙基)-4-(对二乙氨基苯基-六三烯基)二溴化吡啶鎓 (FM 4-64) 作为重要染色剂，以跟踪体膜内化并转运至酵母中的液泡。 30℃处理60分钟后，FM 4-64对液泡膜进行染色（环染色图案）。 FM 4-64 似乎并未通过被动扩散到达液泡，因为在 0 摄氏度时它只对质膜 (PM) 进行染色。将细胞加热至 25°C 后，PM 染色减少，5-10 分钟内细胞质中的小点状结构变得明显。再过20-40分钟后，PM和细胞质点状染色随着液泡膜的染色而消失。在稳态条件下，FM 4-64 染色对液泡膜具有特异性；其他膜结构未染色。该染料作为液泡动力学的敏感报告基因，检测有丝分裂过程中分离结构的形成、液泡裂变/融合事件以及不同类别的液泡蛋白分选（vps）突变体中的液泡形态等事件。在 E 类突变体（例如 vps27）中观察到一个特别引人注目的模式，其中 500-700 nm 细胞器（假定的前液泡区室）被 FM 4-64 强烈染色，而液泡膜呈弱荧光。 FM 4-64 在 15°C 的内化延迟了液泡标记并将 FM 4-64 捕获在 PM 和液泡之间的细胞质中间体中。细胞质中的中间结构很可能是内体，因为它们的染色依赖于温度、时间和能量。有趣的是，与荧光黄摄取不同，FM 4-64 的液泡标记在 sec18、sec14、end3 和 end4 突变体中未被阻断，但在 sec1 突变细胞中被阻断。最后，使用透化酵母原生质球重建 FM 4-64 运输，我们发现 FM 4-64 从内体样中间室（标记为 15°C）到液泡的递送是 ATP 和胞质溶胶依赖性的。因此，我们证明 FM 4-64 是液泡膜的一种新的重要染色剂、内吞中间体的标记物以及用于体外检测内体到液泡膜转运的荧光剂。
We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.