Endocytosis and Membrane Turnover in the Germ Tube of Uromyces fabae

Abstract

We have used the fluorescent dye FM4-64 as a tracer to demonstrate bulk membrane internalization (endocytosis) and redistribution of the dye within the cytoplasm of the germ tube of the rust fungus Uromyces fabae. Staining of the hyphal membrane was detected 4 s after application of FM4-64 and reached a maximum after 1 min. The highest fluorescence intensity occurred in the apex. Subsequently, staining of the plasma membrane decreased and a subapical region of the fungal protoplast (5 20 µm from the tip) displayed increasing fluorescence with a maximum after 5 min. Fluorescence in the subapical region was redistributed to an area in the hyphal tip, which corresponds to the accumulation of apical vesicles, after 10 15 min and subsequently to a cytoplasmic region in front of the two nuclei (35 45 µm from the tip). We conclude from our measurements of membrane fluorescence that the turnover time from endocytosis to secretion of the dye amounts to 15 min. The uptake of the dye into the cytoplasm, but not membrane loading, could be inhibited completely with 5 mM NaN3 or by a temperature shift to 4°C. This is the first evidence for endocytosis in a fungal germ tube.