Tracing the endocytic pathway of Aspergillus nidulans with FM4-64

Simple procedures using FM4-64 to follow membrane internalization and transport to the vacuolar system and endomembranes in Aspergillus nidulans are described. FM4-64 internalization is energy, temperature and F-actin dependent, strongly suggesting that it occurs by endocytosis. The dye sequentially labels: (i) cortical punctuate organelles whose motility resembles that of yeast actin patches; (ii) ∼0.7 μm circular, hollow structures representing mature endosome/vacuole; and (iii) intermediate and large (2-3 μm in diameter) size vacuoles whose lumen is strongly labeled with 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDCFDA). These large vacuoles possibly correspond to the final stage of one branch of the endocytic pathway. In addition, FM4-64 labels strongly the mitochondrial network and weakly the nuclear membrane. A class of cytoplasmic punctuate organelles which become fluorescent very shortly after dye loading and that can move in either apical or basal direction at an average rate of 2-3 μm s−1 is also described. This work provides a useful framework for the phenotypic characterization of A. nidulans mutants affected in endocytosis.