Journal of Microscopy, Vol. 198, Pt 3, June 2000, pp. 246±259. Received 7 January 2000; accepted 3 March 2000 Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae S. FISCHER-PARTON, R. M. PARTON, P. C. HICKEY, J. DIJKSTERHUIS, H. A. ATKINSON & N. D. READ Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, U.K. Key words. Confocal microscopy, endocytosis, FM1-43, FM4-64, fungal hyphae, Spitzenko Èrper, tip growth, TMA-DPH, vesicle trafficking. Summary 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 endocyto- sis. 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, Magna- porthe 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. Introduction Vesicle trafficking is fundamental to numerous activities in eukaryotic organisms, and underlies many of the basic processes involved in cell growth and differentiation. The vesicle trafficking network includes exocytosis and endocy- tosis (Gruenberg & Clague, 1992; Rothman, 1994). In filamentous fungi a reasonable amount is understood about exocytosis, whereas very little is known about endocytosis. Most of our current understanding of vesicle trafficking in filamentous fungi is concerned with tip growth and is based upon ultrastructural studies (e.g. Grove & Bracker, 1970; Howard, 1981), pharmacological treatments (e.g. Howard & Aist, 1980), analyses of mutants (Wu et al., 1998; Seiler et al., 1999) and mathematical modelling of vesicle trafficking in relation to tip growth (e.g. Bartnicki-Garcia et al., 1989). During tip growth, extension of the hypha is confined to a region occupying only a few micrometres at the hyphal apex and involves highly polarized exocytosis. Secretory vesicles deliver membrane, cell wall precursors and wall-building enzymes to the hyphal tip, and many extracellular enzymes released into the surrounding med- ium are also believed to be secreted from this region (reviewed by Wessels, 1993). In higher fungi (i.e. members of the Ascomycota, Basidiomycota and Deuteromycota) vesicle trafficking to the apex during tip growth is highly organized and involves the activity of a specific, multicomponent organelle complex which, in most cases, is called the Spitzenko Èrper ( `apical body'). This structure is predominated by secretory vesicles which make up what is commonly described as an `apical vesicle cluster' (Grove & Bracker, 1970; Lo Âpez-Franco & Bracker, 1996). The dynamic behaviour of the Spitzenko Èr- per has indicated that it is intimately associated with the precise growth pattern of the hyphal apex (Girbardt, 1957; Bartnicki-Garcia et al., 1995; Lo Âpez-Franco & Bracker, 1996). From studies of tip-growing plant cells (Steer & Steer, 1989; Miller et al., 1997) it has been suggested that membrane recycling via endocytosis is a critical part of the process of apical extension. However, reports of the existence of endocytosis in filamentous fungi have been conflicting. Work by Caesar-Ton That et al. (1987) identified a fraction from hyphae of Neurospora crassa that was rich in coated vesicles and possessed a major polypeptide with a molecular weight similar to that of the heavy chain of Correspondence to: Nick Read. Tel: 144 (0)131 650 5335; fax: 144 (0)131 650 5392; e-mail: nick.read@ed.ac.uk 246 q 1999 The Royal Microscopical Society,