Fischer-Parton S, Parton R M, Hickey P C, Dijksterhuis J, Atkinson H A, Read N D
Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, U.K.
J Microsc. 2000 Jun;198(Pt 3):246-59. doi: 10.1046/j.1365-2818.2000.00708.x.
Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used membrane-selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4-64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4-64 was taken up into both the apical and subapical compartments of living hyphae in a time-dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.
两亲性苯乙烯基染料的共聚焦显微镜已被用于研究活真菌菌丝中的内吞作用和囊泡运输。用FM4-64、FM1-43或TMA-DPH处理菌丝，这三种是最常用的膜选择性染料，被报道为内吞作用的标志物。所有这三种染料都能在菌丝内迅速内化。由于其染色模式、更高的光稳定性和低细胞毒性，发现FM4-64最适合对生长中的菌丝尖端内顶端囊泡簇的大小、形态和位置的动态变化进行成像。FM4-64以时间依赖性方式被摄取到活菌丝的顶端和亚顶端区室中。在所测试的一系列真菌物种（构巢曲霉、灰葡萄孢、稻瘟病菌、粗糙脉孢菌、布氏毛霉、禾柄锈菌、立枯丝核菌、核盘菌和绿色木霉）中，染色分布模式大致相似。随着时间的推移，FM4-64从质膜内化，出现在与假定的内体、顶端囊泡簇、液泡膜和线粒体相对应的结构中。这些观察结果与通过内吞作用进行的染料内化一致。本文提出了一个生长中菌丝内囊泡运输网络的推测模型。
