Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae

Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were t reated with FM4-64, FM1-43 or TMA-DPH, three of the most commonly used memb rane-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 clus ter within growing hyphal tips because of its staining pattern, greater pho tostability 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 spe cies tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Ne urospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia s olani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4-64 was internalized from the plasma membrane appearing in structures correspon ding to putative endosomes, the apical vesicle cluster, the vacuolar membra ne and mitochondria. These observations are consistent with dye internaliza tion by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.