S. Fischer-parton et al., Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae, J MICROSC O, 198, 2000, pp. 246-259
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.