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) approximately 0.7 microm circular, hollow structures representing mature endosome/vacuole; and (iii) intermediate and large (2-3 microm 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 microm s(-1) is also described. This work provides a useful framework for the phenotypic characterization of A. nidulans mutants affected in endocytosis.
Peñalva
Miguel A
MA
Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain. penalva@cib.csic.es
D001233:Aspergillus nidulans / Q000502:physiology*; D004705:Endocytosis; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D009685:Nuclear Envelope / Q000378:metabolism; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D010784:Photomicrography; D011726:Pyridinium Compounds / Q000378:metabolism* / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism* / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods
C109098:2,7-dihydrodichlorofluorescein diacetate; C092350:FM 4-64; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds